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Originally published In Press as doi:10.1074/jbc.M412006200 on November 4, 2004

J. Biol. Chem., Vol. 280, Issue 2, 1156-1164, January 14, 2005
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A Role for Rat Inositol Polyphosphate Kinases rIPK2 and rIPK1 in Inositol Pentakisphosphate and Inositol Hexakisphosphate Production in Rat-1 Cells*

Makoto Fujii and John D. York{ddagger}

From the Departments of Pharmacology and Cancer Biology and of Biochemistry, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, October 22, 2004 , and in revised form, November 3, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Over 30 inositol polyphosphates are known to exist in mammalian cells; however, the majority of them have uncharacterized functions. In this study we investigated the molecular basis of synthesis of highly phosphorylated inositol polyphosphates (such as inositol tetrakisphosphate, inositol pentakisphosphate (IP5), and inositol hexakisphosphate (IP6)) in rat cells. We report that heterologous expression of rat inositol polyphosphate kinases rIPK2, a dual specificity inositol trisphosphate/inositol tetrakisphosphate kinase, and rIPK1, an IP5 2-kinase, were sufficient to recapitulate IP6 synthesis from inositol 1,4,5-trisphosphate in mutant yeast cells. Overexpression of rIPK2 in Rat-1 cells increased inositol 1,3,4,5,6-pentakisphosphate (I(1,3,4,5,6)P5) levels about 2–3-fold compared with control. Likewise in Rat-1 cells, overexpression of rIPK1 was capable of completely converting I(1,3,4,5,6)P5 to IP6. Simultaneous overexpression of both rIPK2 and rIPK1 in Rat-1 cells increased both IP5 and IP6 levels. To reduce IPK2 activity in Rat-1 cells, we introduced vector-based short interference RNA against rIPK2. Cells harboring the short interference RNA had a 90% reduction of mRNA levels and a 75% decrease of I(1,3,4,5,6)P5. These data confirm the involvement of IPK2 and IPK1 in the conversion of inositol 1,4,5-trisphosphate to IP6 in rat cells. Furthermore these data suggest that rIPK2 and rIPK1 act as key determining steps in production of IP5 and IP6, respectively. The ability to modulate the intracellular inositol polyphosphate levels by altering IPK2 and IPK1 expression in rat cells will provide powerful tools to study the roles of I(1,3,4,5,6)P5 and IP6 in cell signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Most of the over 30 inositol polyphosphates present in mammalian cells have unknown physiological functions (1–3). One of the well characterized inositol polyphosphates is a second messenger, inositol 1,4,5-trisphosphate (I(1,4,5)P3),1 which participates in intracellular Ca2+ mobilization (4) and also serves as a precursor of highly phosphorylated inositol polyphosphates such as inositol tetrakisphosphate (IP4), inositol pentakisphosphate (IP5), and inositol hexakisphosphate (IP6) (1–3). Recently the inositol polyphosphate synthetic pathway in budding yeast, Saccharomyces cerevisiae, has been identified (5–8). In yeast, I(1,4,5)P3, which is hydrolyzed from phosphatidylinositol 4,5-bisphosphate by phospholipase C, is sequentially phosphorylated to IP6 by two inositol polyphosphate kinases, a multiple specificity IP3/IP4 kinase (inositol polyphosphate kinase 2 (Ipk2)) and an IP5 2-kinase (inositol polyphosphate kinase 1 (Ipk1)). On the other hand, a synthetic pathway of IP6 from I(1,4,5)P3 in mammalian cells is thought to be more complex and to be a sequence of I(1,4,5)P3 -> I(1,3,4,5)P4 -> I(1,3,4)P3 -> I(1,3,4,6)P4 -> I(1,3,4,5,6)P5 -> IP6 (2, 3). In this pathway, IP3 3-kinases (2), inositol polyphosphate 5-phosphatases (9), and I(1,3,4)P3 5/6-kinase (3) appear to catalyze the first three steps to produce I(1,3,4,6)P4, respectively. Additionally the mammalian orthologs of IPK2 (also known as inositol polyphosphate multikinase) and IPK1 were cloned recently by several independent groups and partially characterized in vitro (10–13). These two kinases possibly catalyze the last two steps, respectively; however, the possibility remains that IP6 is synthesized from the sequential phosphorylation of I(1,4,5)P3 by IPK2 and IPK1 in mammalian cells as it is in yeast cells. Currently there is no definitive evidence describing the inositol polyphosphate synthetic pathways in mammalian cells (2, 3).

Several recent works suggest possible functions of highly phosphorylated inositol polyphosphates and/or their kinases. For example, IP5 has been shown to modulate human immunodeficiency virus, type 1, Gag protein assembly (14), and IP6 has been reported to bind the clathrin assembly proteins AP2 and AP3 thus inhibiting clathrin cage assembly in vitro (15–18). Additionally IP6 stimulates non-homologous DNA end joining of double strand DNA breaks by binding to the Ku70/80 subunits of DNA-dependent protein kinase in vitro (19–21). Yeast genetic data also suggest that these kinases and/or their inositol polyphosphate products are involved in transcription of specific genes, mRNA export, and chromatin remodeling (5–7, 22–24). However, most of the data mentioned above were determined in vitro or in yeast, and evidence in mammalian cells is still lacking. Additionally some of the data obtained from in vitro studies may be experimental artifacts due to the effect of the highly polarized negative charge of these inositol polyphosphate molecules (25).

To address the synthetic pathway and the function of inositol polyphosphates in mammalian cells, it is important to establish a model system whereby the inositol polyphosphate levels can be modulated in vivo. In this study, we analyzed the effects of either overexpression or knock-down of inositol polyphosphate kinase(s) on inositol polyphosphate levels in Rat-1 cells. We showed the accumulation of I(1,3,4,5,6)P5 and IP6 by overexpression of IPK2 and IPK1. We also succeeded in decreasing cellular I(1,3,4,5,6)P5 levels by RNA interference (RNAi). These data reveal the involvement of IPK2 and IPK1 in I(1,3,4,5,6)P5 and IP6 synthesis in vivo. Furthermore the ability to modulate the intracellular inositol polyphosphate levels shown here by altering IPK2 and IPK1 expression in rat cells will provide powerful tools to study the roles of I(1,3,4,5,6)P5 and IP6 in eukaryotic cell signaling.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfection—Phoenix and Rat-1 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% CO2. Culture medium was supplemented with 100 units/ml penicillin G and 100 µg/ml streptomycin. The cell lines expressing tetracycline-inducible constructs were maintained in the same condition except using 10% Tet system-approved FBS (Clontech). Retroviruses were produced by transient transfection of the plasmid into phoenix cells by the calcium phosphate method as described elsewhere (26). Rat-1 cells were infected with retrovirus-containing media in the presence of 8 µg/ml hexadimethrine bromide (Sigma) for overnight. Thirty-six hours later, cells were selected in the presence of appropriate antibiotics for at least 2 weeks and then used for experiments. The concentration of antibiotics used for selection were: Geneticin, 800 µg/ml; hygromycin, 400 µg/ml; and puromycin, 1.5 µg/ml, respectively.

RNA Preparation and Cloning of Rat IPK2 and Rat IPK1 by RT-PCR—Total RNA was isolated according to the manufacturer's recommendations using the RNeasy minikit (Qiagen). Briefly 1 x 107 Rat-1 cells were lysed into Buffer RLT with 1% 2-mercaptoethanol and then applied to the RNeasy minicolumn. After washing the column, total RNA was eluted with 40 µl of RNase-free water. RT-PCR was performed using the RobusT I RT-PCR kit (Finnzymes). Reverse transcription of 1 µg of RNA was performed at 42 °C for 1 h in 50 µl of reaction mixture containing 1x RobusT I reaction buffer, 1.5 mM MgCl2, 40 units of RNase inhibitor, 0.8 mM dNTPs, 0.2 µM oligo(dT), 0.2 µM sense and antisense primers, 5 units of avian myeloblastosis virus reverse transcriptase, and 1 unit of DyNAzyme EXT DNA polymerase. The subsequent PCR was done at 94 °C for an initial 2 min followed by 25 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min and final extension at 72 °C for 10 min. The primers were designed to amplify the entire open reading frame of the rat ortholog of IPK2 (rIPK2) (GenBankTM accession number AY014898 [GenBank] ) (10). The primers for the rat ortholog of IPK1 (rIPK1) were designed based on the mouse IPK1 ortholog (GenBankTM accession number XM_283126). The primers used for rIPK2 were: sense, 5'-TTT TTG TCG ACC ACC ATG GCC GCC GAG CCC CCA-3' (the SalI site is underlined); and antisense, 5'-TTT TTG ATA TCG ATT CAA CTG TCC AAG ATA CTC CG-3' (the ClaI site is underlined). The primers used for rIPK1 were: sense, 5'-AAC TCG AGC ACC ACC ATG GAA GAG GGG AAA AT-3' (the XhoI site is underlined); and antisense, 5'-AAT CTA GAA AGC TTT TAG ACC TTA TGG AGA ACT AAT GTG CCC G-3' (the HindIII site is underlined). The RT-PCR products were cloned into pCR2.1 TA cloning vector (Invitrogen). The substitution of Asp-127 to Ala in rIPK2 (rIPK2 D127A) was performed by PCR with the following mutagenic primers (the D127A mutation is underlined): sense, 5'-AAG CCC TGT ATA ATG GCC GTG AAG ATT GGG CGG-3'; and antisense, 5'-CCG CCC AAT CTT CAC GGC CAT TAT ACA GGG CTT-3'. All constructs were sequenced by the ABI Prism Big Dye Terminator Ready reaction kit (Applied Biosystems).

Plasmid Construction—The SalI/EcoRI fragment of pCR2.1/rIPK2 (and also the D127A mutant) or the XhoI/EcoRI fragment of pCR2.1/rIPK1 were inserted into the SalI/EcoRI sites of pGST4 (27) to produce pGST/rIPK2 or pGST/rIPK1, respectively. The EcoRI fragments of pCR2.1/rIPK2 or pCR2.1/rIPK1 were inserted into the same site of pUNI10 (28) and then recombined with pRS426-CUP1-MYC3-LOXP (29) using the cre-loxP system (28) to produce the yeast expression vector for rIPK2 or rIPK1, respectively. To introduce the N-terminal GFP or MYC tag to the tetracycline-induced gene expression vector pRevTRE (Clontech), the sequence for GFP or Myc tag was amplified by PCR using phGFP105-C1 (30) or pRS426-CUP1-MYC3-LOXP (29) as template. The primers were: GFP sense, 5'-TTT TTG GAT CCA CCA TGG TGA GCA AGG GC-3' (the BamHI site is underlined); GFP antisense, 5'-TTT TTG TCG ACT TGT ACA GCT CGT CCA TGC C-3' (the SalI site is underlined); MYC sense, 5'-TTT TTG GAT CCA CCA TGG GAT TCG AGC TAT GCG GC-3' (the BamHI site is underlined); and myc antisense, 5'-TTT TTG TCG ACC CTT CGA GAC TAG TGC GGC-3' (the SalI site is underlined), respectively. The PCR products were digested by BamHI/SalI and inserted into the same sites of pRevTRE to produce pRevTRE/GFP or pRevTRE/myc, respectively. The NheI/HindIII fragment of pEGFP-C1 (Clontech) was also cloned into the same sites of pTRE2 (Clontech) to produce pTRE/GFP. The SalI/ClaI fragment of pCR2.1/rIPK2 was inserted into the same sites of pRevTRE/GFP to produce pRevTRE/GFP-rIPK2. The XhoI/HindIII fragment of pCR2.1/rIPK1 was inserted into the SalI/HindIII sites of pRevTRE/GFP or pRevTRE/myc for pRevTRE/GFP-rIPK1 or pRevTRE/myc-rIPK1, respectively. The BamHI/{Delta}ClaI fragment of pRevTRE/GFP-rIPK2 or pRevTRE/GFP-rIPK1 was inserted into the BamHI/SnaBI sites of pBabePuro (31) to make pBabePuro/GFP-rIPK2 or pBabePuro/GFP-rIPK1, respectively. The BamHI/SalI fragment of pTRE/GFP was inserted into the same site of pBabePuro to produce pBabePuro/GFP. S. cerevisiae IPK2 (and also kin–) (6) or IPK1 (5) was cloned by PCR using pUNI/ScIPK2 (kin–) or genomic DNA as template, respectively. The primers were: ScIPK2 sense, 5'-ACT CTA TAA AGC TTT CTA TAA AAT GGA TAC GGT AAA CAA TTA TAG G-3' (the HindIII site is underlined); ScIPK2 antisense, 5'-ACT CTA GTC GAC AAG ACA AGG TAA ACT TCA CCT CTC A-3' (the SalI site is underlined); ScIPK1 sense, 5'-TTT TTG TCG ACC ACC ATG CAA GTC ATC GGA CGT GG-3' (the SalI site is underlined); and ScIPK1 antisense, 5'-TTT TTT CTA GAA AGC TTC TGC CAG TAC CAA AGG TGG-3' (the HindIII site is underlined), respectively. The PCR product of ScIPK2 was digested by HindIII/SalI site and cloned into the same site of pTRE/GFP to produce pTRE/GFP-ScIPK2. The BamHI/SalI fragment of pTRE/GFP-ScIPK2 was cloned into the same sites of pRevTRE to make pRevTRE/GFP-ScIPK2. The PCR product of ScIPK1 was digested by SalI/HindIII and cloned into the same sites of pRevTRE to make pRevTRE/GFP-ScIPK1. For the vector-based rIPK2 siRNA construct, pSUPER/rIPK2–3, which targeted nucleotides 1075–1093 of rIPK2, synthetic DNA primers (sense, 5'-GAT CCC CGC GGA AGT GCG GAT GAT AGT TCA AGA GAC TAT CAT CCG CAC TTC CGC TTT TTG GAA A-3'; and antisense, 5'-AGC TTT TCC AAA AAG CGG AAG TGC GGA TGA TAG TCT CTT GAA CTA TCA TCC GCA CTT CCG CGG G-3') were annealed and then cloned into BglII/HindIII sites of pSUPER (32). All constructs were sequenced by the ABI Prism Big Dye Terminator Ready reaction kit (Applied Biosystems).

Expression and Purification of Recombinant GST Fusion Protein— The expression and purification of GST-rIPK2 and GST-rIPK1 were performed as described previously (29) with minor modifications. Briefly competent BL21 Escherichia coli cells were transformed by pGST/rIPK2 or pGST/rIPK1. One liter of LB was cultured at 37 °C to an absorbance of 0.8–1.0 at 600 nm. Expression was induced by the addition of 0.1 mM isopropyl {beta}-D-thiogalactopyranoside (final concentration). The cells were grown for 20 h at 20 °C, harvested by centrifugation at 4 °C, and resuspended in 20 ml of ice-cold phosphate-buffered saline containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride and one CompleteTM Mini protease inhibitor mixture tablet (Roche Applied Science)/10 ml of buffer). The cells were lysed by five passages through a cell cracker (a high shear fluid-processing system for cell rupture, Microfluidics Corp.). Triton X-100 was added to lysates at final concentrations of 1%, and then lysates were subjected to centrifugation at 15,000 x g for 15 min at 4 °C. The supernatant was then incubated with 1 ml of 50% glutathione-Sepharose slurry (Amersham Biosciences) for 1 h at 4 °C with shaking. The Sepharose was washed three times with 15 ml of ice-cold phosphate-buffered saline, and the GST fusion proteins were eluted from the Sepharose with 1 ml of 10 mM reduced glutathione in 50 mM Tris-HCl (pH 8.0) and stored at –80 °C in aliquots.

Inositol Phosphate Kinase Assays—To synthesize [3H]I(1,3,4,5,6)P5, [3H]I(1,4,5)P3 was phosphorylated by 100 ng of recombinant Arabidopsis thaliana ortholog of Ipk2 (AtIpk2{beta}), which phosphorylates I(1,4,5)P3 to I(1,3,4,5,6)P5 (29), for 30 min at 37 °C in the same buffer as that use in the kinase assay (see below), and the reaction was terminated by incubation for 3 min at 95 °C. The inositol phosphate kinase assay performed in vitro was described previously (29). Briefly 10–100 ng of purified recombinant GST-rIPK2 or GST-rIPK1 were incubated independently with 1–10 µM [3H]inositol, [3H]I(1)P, [3H]I(1,4)P2, [3H]I(1,3,4)P3, [3H]I(1,4,5)P3, [3H]I(1,3,4,5)P4, or [3H]I(1,3,4,5,6)P5 for 30 min at 37 °C in buffer containing 2 mM ATP, 50 mM Hepes-NaOH (pH 7.5), 50 mM KCl, 10 mM MgCl2, 10 mM phosphocreatine, and 0.2 units/µl phosphocreatine kinase. To determine whether the IP4 product of I(1,4,5)P3 phosphorylation was I(1,3,4,5)P4 or I(1,4,5,6)P4, the reaction was terminated by incubation for 3 min at 95 °C and then dispensed in half. One half of the reaction was incubated with 100 ng of purified recombinant type I inositol polyphosphate 5-phosphatase (9) for another 30 min at 37 °C. To determine whether the IP5 product of I(1,4,5)P3 phosphorylation was I(1,3,4,5,6)P5, half of the reaction was incubated with 100 ng of purified recombinant GST-AtIpk1, GST-fused A. thaliana ortholog of I(1,3,4,5,6)P5 2-kinase,2 for another 30 min at 37 °C. To test whether rIPK2 or rIPK1 phosphorylated a range of different inositol polyphosphates, 100 ng of GST-rIPK2 or GST-rIPK1 were incubated independently with 10 µM I(1,4,5)P3, I(1,3,4,5)P4, I(1,3,4,6)P4, I(1,4,5,6)P4, or I(1,3,4,5,6)P5 in 50 mM Hepes-NaOH (pH 7.5), 50 mM KCl, 10 mM MgCl2, and a trace amount of [{gamma}-32P]ATP (200,000 cpm) for 30 min at 37 °C. All reactions were terminated by the addition of 30 times the reaction volume of 10 mM NH4H2PO4 (pH 3.5) and analyzed by HPLC as described previously (5).

Cell Lysate Preparation and Western Blotting Analysis—2 x 105 Rat-1 cells stably expressing the indicated plasmid were seeded in 60-mm culture dishes containing 5 ml of the appropriate media with or without 2 µg/ml doxycycline and cultured for 2–3 days. Cells were washed twice with 5 ml of ice-cold phosphate-buffered saline and then extracted with 200 µl of ice-cold 1% Nonidet P-40 TNE buffer (10 mM Tris-HCl (pH 7.6), 1% Nonidet P-40, 150 mM NaCl, 2 mM phenylmethylsulfonyl fluoride, and one CompleteTM Mini protease inhibitor mixture tablet/10 ml of buffer). After a 15-min incubation on ice, the lysate was recovered by centrifugation (14,000 rpm for 15 min at 4 °C). The supernatants were subjected to protein assay using bovine serum albumin as standard. One microgram of cell lysates was separated by SDS-PAGE. Immunostaining of gels was carried out using an ECL Western blotting detection system (Amersham Biosciences) using a rabbit polyclonal anti-GFP antibody (Clontech).

Fluorescence Microscopy—Rat-1 cells stably expressing GFP fusion constructs were washed with prewarmed Hanks' balanced salt solution and observed under the Olympus IX70 inverted microscope equipped with a confocal head using a LCPlan Fl 40x/0.60 numerical aperture lens. Images were recorded using UltraVIEWTM imaging software (PerkinElmer Life Sciences).

myo-[3H]Inositol Labeling, Isolation, and Analysis of Soluble Inositol Phosphates—S. cerevisiae cells carrying expression plasmid were grown in 1 ml of complete minimal medium lacking uracil with 100 µM CuSO4 and 40 µCi/ml myo-[2-3H]inositol to late logarithmic phase. The cells were harvested, and the soluble lysate containing the inositol polyphosphates was isolated according to York et al. (5).

1 x 105 Rat-1 cells stably expressing the indicated plasmid were seeded into a 60-mm culture dish containing 3 ml of appropriate media. One day after incubation, cells were washed twice with 5 ml of prewarmed Medium-199 and then labeled with 10–20 µCi/ml myo-[2-3H]inositol for 2 days in 2 ml of Medium-199 supplemented with dialyzed 10% FBS with or without 2 µg/ml doxycycline. Cells were washed twice with ice-cold phosphate-buffered saline and then harvested in 500 µl of 1 N HCl. The soluble fraction containing the inositol polyphosphates was isolated as mentioned above. To determine whether the IP5 product in the rIPK2-expressing Rat-1 cells was I(1,3,4,5,6)P5, the lysate was dried using a SpeedVac and resuspended to 50 mM Hepes-NaOH (pH 7.5). The lysate was incubated with 100 ng of purified recombinant GST-AtIpk1 for 30 min at 37 °C.

Northern Blotting Analysis—Northern blot analysis was performed as described by Stevenson et al. (33). The entire open reading frame of each gene was used as probe and labeled using Ready-To-GoTM DNA labeling beads (Amersham Biosciences) with [{alpha}-32P]dCTP. Hybridization was carried out using ExpressHyb hybridization solution (Clontech) following the manufacturer's recommendations.

Colony Formation Assay in Soft Agar—5 x 104 Rat-1 cells stably expressing the indicated plasmid were seeded into a 35-mm culture dish containing 1 ml of culture media with 0.4% agar. The dishes had been coated with 2 ml of culture media containing 0.7% agar. Cells were fed with 0.5 ml of culture media containing 0.4% agar once a week. Colony formation was scored after 3 weeks at 37 °C in a humidified atmosphere of 5% CO2.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of Rat IPK2 and Rat IPK1 Orthologs—To investigate the function of inositol polyphosphates in mammalian cells, we first cloned rat orthologs of IPK2 and IPK1. Saiardi et al. (10) have published the cloning of rIPK2, so we used this sequence information for cloning (see "Experimental Procedures").

When we started this project, the DNA sequence information of rIPK1 was not available. However, we could use both the human and mouse sequences of IPK1. These two orthologs possess about 88% identity at the DNA level. Based on this information, we designed cloning primers for rIPK1 and performed RT-PCR (see "Experimental Procedures"). The rat ortholog of IPK1 possesses 91.9 and 96.3% of amino acid identity to the human and mouse orthologs, respectively. The rIPK1 contains the conserved motifs EXKPK, CRXC, (F/Y)CPLDL, and D(L/V)DLK(P/S)X(E/M) (13), the function of which are still unknown. Recently the predicted open reading frame of rIPK1 was published in GenBankTM (GenBankTM accession number XM_225201). This prediction was based on GenomeScan analysis of the rat genomic sequence (GenBankTM accession number NW_047490). Our cDNA differed somewhat from the predicted intron/exon splicing identified by the GenomeScan software. Therefore, we submitted a new sequence, and GenBankTM issued a new accession number (GenBankTM accession number AY823319 [GenBank] ) for rIPK1.

Characterization of Rat IPK2 and Rat IPK1 in Vitro—The cloning and characterization of rat and human orthologs of IPK2 have been reported by several independent groups (10–12). However, there is controversy about the substrate specificity among these reports. Therefore, we analyzed the rIPK2 in vitro. We incubated GST-rIPK2 with [3H]I(1,4,5)P3 for 30 min and then analyzed the products by HPLC. Fig. 1A shows the elution pattern of the substrate [3H]I(1,4,5)P3. 10 ng of GST-rIPK2 phosphorylated I(1,4,5)P3 to IP4 and IP5 (Fig. 1B). It was reported that both rat and human orthologs of IPK2 prefer to phosphorylate the D-3 position of I(1,4,5)P3, although these data were based on the comparison of the elution position of products to standards on HPLC and lacked biochemical evidence (10, 11). Therefore, we identified these IP4 and IP5 isomers using enzymatic treatment. To identify the IP4 isomer, we treated the reaction product with recombinant type I inositol polyphosphate 5-phosphatase (9). The inositol polyphosphate 5-phosphatase dephosphorylated I(1,3,4,5)P4 to I(1,3,4)P3 but did not dephosphorylate I(1,4,5,6)P4. Upon inositol polyphosphate 5-phosphatase treatment, the IP4 was completely dephosphorylated to I(1,3,4)P3 (Fig. 1C). These data show that the IP4 intermediate is I(1,3,4,5)P4. Next we attempted to identify the IP5 isomer generated by rIPK2. To do this, we treated the reaction products with AtIpk1, the A. thaliana ortholog of I(1,3,4,5,6)P5 2-kinase (the gift of Dr. Stevenson-Paulik, York laboratory). The AtIpk1 phosphorylates the D-2 position of I(1,3,4,5,6)P5 to produce IP6. Upon AtIpk1 treatment, the IP5 was completely phosphorylated to IP6 (Fig. 1D). These data demonstrate that the IP5 isomer is I(1,3,4,5,6)P5. Taken together, the rIPK2 phosphorylates I(1,4,5)P3 to I(1,3,4,5,6)P5 through I(1,3,4,5)P4 in vitro. This activity differs from yeast Ipk2, which prefers to phosphorylate the D-6 position of I(1,4,5)P3 and then the D-3 position to produce I(1,3,4,5,6)P5 (6, 7).



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  		FIG. 1.
Kinase assay of rat IPK2 and rat IPK1 in vitro. A, HPLC trace of substrate [3H]I(1,4,5)P3. B, 10 ng of GST-rIPK2 was incubated with 10 µM [3H]I(1,4,5)P3 for 30 min at 37 °C. C and D, to identify the inositol polyphosphate isomers, GST-rIPK2 phosphorylation products (B) were treated with 100 ng of type I inositol polyphosphate 5-phophatase, which hydrolyzes I(1,3,4,5)P4 but not I(1,4,5,6)P4 (C), or 100 ng of GST-AtIpk1, which phosphorylates I(1,3,4,5,6)P5 to IP6 (D), for 30 min at 37 °C, respectively. E, HPLC trace of substrate [3H]I(1,3,4,5,6)P5. F, 100 ng of GST-rIPK1 was incubated with 10 µM [3H]I(1,3,4,5,6)P5 for 30 min at 37 °C. The substrate and phosphorylation products were separated by HPLC using the Partisphere strong anion exchange column as described under "Experimental Procedures." The elution position of each inositol polyphosphate isomer is indicated by the arrow.

 
To produce the kinase-negative mutant of rIPK2, we changed Asp-127 to Ala (rIPK2 D127A) by site-directed mutagenesis; this mutant corresponds to the previously described D131A mutant of ScIpk2 (Scipk2 kin–) (6). The Scipk2 kin– is completely kinase-inactive, but the rIPK2 D127A mutant retains roughly 1% of the wild type kinase activity toward I(1,4,5)P3 (data not shown). We also tested the substrate specificity of rIPK2 using different inositol polyphosphates because it is known that the rat and human orthologs are able to phosphorylate several different inositol polyphosphate isomers (10–12). Among the substrates we used, 100 ng of rIPK2 phosphorylated I(1,4,5)P3, I(1,3,4,5)P4, I(1,3,4,6)P4, and I(1,4,5,6)P4 to I(1,3,4,5,6)P5 but not inositol, I(1)P, I(1,3,4)P3, and I(1,3,4,5,6)P5. 100 ng of rIPK2 partially phosphorylated I(1,4)P2 to an unidentified IP3 (data not shown).

Next we characterized the kinase activity and substrate specificity of rIPK1 in vitro. 100 ng of rIPK1 only phosphorylated I(1,3,4,5,6)P5 to IP6 (Fig. 1F) and no other inositol polyphosphates we tested (data not shown). These data are consistent with the recently reported characterization of human IPK1 (13). Fig. 1E shows the elution of substrate [3H]I(1,3,4,5,6)P5.

Complementation of Inositol Polyphosphate Production in Mutant Yeast Cells—We tested whether rIPK2 could rescue inositol polyphosphate synthesis in yeast cells lacking endogenous IPK2. In this experiment, the wild type, ipk2 null (ipk2{Delta}), and ipk2{Delta} strain expressing either rIPK2 or rIPK2 D127A mutant were metabolically labeled with [3H]inositol, and then the soluble inositol polyphosphates were analyzed by HPLC. In the wild type strain, IP6 was the major inositol polyphosphate (Fig. 2A). In the ipk2{Delta} strain, IP3 accumulated because Ipk2 is the major kinase responsible for the conversion of IP3 to produce higher inositol polyphosphates such as IP5 and IP6 (Fig. 2B) (6, 7). The ipk2{Delta} strain harboring the rIPK2 expression plasmid complemented IP5 and IP6 production (Fig. 2C). These data confirmed that rIPK2 is active in yeast and is capable of converting I(1,4,5)P3 to I(1,3,4,5,6)P5. Expression of the rIPK2 D127A mutant partially rescued IP6 synthesis (Fig. 2D), consistent with our biochemical studies indicating that this mutation in the rat enzyme still possesses weak activity.



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  		FIG. 2.
Complementation of IP6 synthesis by rat IPK2 or rat IPK1 in S. cerevisiae lacking the IPK2 or IPK1 gene, respectively. S. cerevisiae wild type, W303 strain (A), ipk2 null strain (B), ipk2 null strain expressing wild type rIPK2 (C) or rIPK2 D127A mutant (D), ipk1 null strain (E), or ipk1 null strain expressing wild type rIPK1 (F) were grown to late logarithmic phase in complete minimal medium lacking uracil with 100 µM CuSO4 and 40 µCi/ml [3H]inositol. The soluble inositol polyphosphates were extracted and then analyzed by HPLC using the Partisphere strong anion exchange column as described under "Experimental Procedures." The elution positions of inositol polyphosphates were compared with the elution of known species and are indicated by the arrows. Ins, inositol; PP-IP4, diphosphorylinositol tetrakisphosphate; IP2x, inositol bisphosphate.

 
We next tested whether rIPK1 could rescue IP6 synthesis in mutant yeast cells. The yeast strain lacking IPK1 (ipk1{Delta}) accumulated IP5 and a pyrophosphorylated inositol polyphosphate, diphosphorylinositol tetrakisphosphate (Fig. 2E, PP-IP4) (5). Overexpression of rIPK1 complemented this phenotype (Fig. 2F) indicating that the rIPK1 ortholog is also active in yeast.

Expression and Intracellular Localization of Inositol Polyphosphate Kinase(s) in Rat-1 Cells—After confirmation of the enzymatic activity of rIPK2 and rIPK1 in vitro and in budding yeast, we expressed these genes in rat cells. An inducible mammalian expression system (Tet-OnTM gene expression system, Clontech) was chosen thereby enabling the control of protein expression by the tetracycline or tetracycline analogue doxycycline (34). We expressed control GFP, yeast or rat IPK2, and yeast or rat IPK1 as GFP fusion proteins under the control of doxycycline in the Rat-1 rat embryonic fibroblast cells. Appropriate protein expression was confirmed using an anti-GFP immunoblot (data not shown). In the stable cell lines used in the absence of doxycycline, there was some leaky expression of protein, but 2 µg/ml doxycycline induced expression levels of all of the fusion proteins (data not shown).

We investigated the intracellular localization of these proteins. The expression of the GFP fusion proteins was induced by 2 µg/ml doxycycline, and living cells were observed under confocal microscopy. In contrast to the ubiquitous localization of GFP alone (Fig. 3A), GFP-rIPK2 was localized in the nucleus (Fig. 3C). To our knowledge, there is no other report describing rIPK2 localization, but it has been reported that the human IPK2 localizes to the nucleus, and the nuclear localization signal sequence was identified (11). This sequence is conserved between human and rat IPK2, so this nuclear localization signal sequence is likely also functional in rIPK2. It was reported that yeast Ipk2 is localized to the nucleus in yeast cells (6), but when we expressed yeast Ipk2 in Rat-1 cells, it localized to the cytosol (Fig. 3B). This difference probably reflects the difference of nuclear localization mechanisms between yeast and Rat-1 cells. Yeast Ipk1 is localized to the nuclear envelope in yeast cells (5), but both yeast Ipk1 and rat IPK1 were localized ubiquitously in Rat-1 cells (Fig. 3, D and E).



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  		FIG. 3.
Intracellular localization of GFP-fused inositol polyphosphate kinase(s) in Rat-1 cells. Rat-1 cells stably expressing pRevTRE/GFP (A), pRevTRE/GFP-ScIPK2 (B), pRevTRE/GFP-rIPK2 (C), pRevTRE/GFP-ScIPK1 (D), or pRevTRE/GFP-rIPK1 (E) were grown under normal growth conditions with 2 µg/ml doxycycline for 2 days to induce protein expression. Cells were washed with prewarmed Hanks' balanced salt solution and observed by fluorescence microscopy as indicated under "Experimental Procedures."

 
Inositol Polyphosphate Profiles in IPK2- or IPK1-overexpressing Rat-1 Cells—To address whether the overexpression of these genes effects inositol polyphosphate production, we labeled these cells using [3H]inositol and then analyzed the soluble inositol polyphosphates by HPLC. In the untransfected (Fig. 4A) or GFP-expressing Rat-1 cells (Fig. 4B), IP5 was the major inositol polyphosphate, and it was about 16–18% of the total inositol polyphosphates. IP6 was about 7–8% of total inositol polyphosphates in these cells. Both the yeast (Fig. 4C) and rat IPK2 overexpression (Fig. 4E) increased IP5 levels about 2–3-fold compared with control cells. Overexpression of Scipk2 kin– (Fig. 4D) did not show any increase of IP5 levels, but the rIPK2 D127A mutant (Fig. 4F) expressing Rat-1 cells showed a subtle but consistent increase in IP5 content compared with control cells. Our data indicate that increased IPK2 activity, nuclear in the case of rIPK2 or cytoplasmic in the case of scIpk2, is sufficient to elevate IP5 levels.



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  		FIG. 4.
Inositol polyphosphate profiles of Rat-1 cells expressing inositol polyphosphate kinase(s). Approximately 1 x 105 Rat-1 cells stably expressing mock plasmid pRevTet-On (A), pRevTRE/GFP (B), pRevTRE/GFP-ScIPK2 (C), pRevTRE/GFP-ScIPK2 D131A (kin–) (D), pRevTRE/GFP-rIPK2 (E), pRevTRE/GFP-rIPK2 D127A (F), pRevTRE/GFP-ScIPK1 (G), or pRevTRE/GFP-rIPK1 (H) were seeded into a 60-mm culture dish containing 3 ml of culture media. One day after growth, cells were labeled in Medium-199 supplemented with 10% dialyzed FBS and containing 20 µCi/ml [3H]inositol for 2 days. 2 µg/ml doxycycline was added to the media to induce protein expression for the same period. The soluble inositol polyphosphates were extracted and then analyzed by HPLC using the Partisphere strong anion exchange column as described under "Experimental Procedures." The elution positions of inositol polyphosphates were compared with the elution of known species and are indicated by the arrows. Ins, inositol.

 
To confirm that the IP5 isomer that accumulates in the IPK2 overexpressing Rat-1 cells is indeed I(1,3,4,5,6)P5, we prepared soluble radiolabeled inositol polyphosphate extracts from these cell lines and subjected the IPs to enzyme analysis. As mentioned above, AtIpk1 can phosphorylate I(1,3,4,5,6)P5 to IP6 but not other IP5 species (data not shown). The IP5 peak in the extract of cells expressing either GFP control or GFP-rIPK2 was completely phosphorylated to IP6 by AtIpk1 in vitro (data not shown). These data show that rIPK2 synthesizes I(1,3,4,5,6)P5 in Rat-1 cells and also shows that the major IP5 isomer in the Rat-1 cells is I(1,3,4,5,6)P5, which is consistent with previous reports (35).

Next we investigated the effect of IPK1 overexpression on inositol polyphosphate production in the Rat-1 cells. Both the yeast Ipk1 and rat IPK1 overexpression increased the IP6 level to about 20% of total inositol polyphosphates (Fig. 4, G and H). This was about a 2.5-fold elevation of IP6 levels compared with control cells. Interestingly the I(1,3,4,5,6)P5 levels were decreased to undetectable levels in these cells, indicating that levels of endogenous rIPK1 are highly regulated and/or rate determining.

Since all of the I(1,3,4,5,6)P5 was converted to IP6 when the cells were overexpressing IPK1 and since I(1,3,4,5,6)P5 was increased with IPK2 overexpression, we attempted to generate high levels of IP6 in Rat-1 cells by overexpressing IPK2 and IPK1 simultaneously. Fig. 5, A and C, shows the HPLC profiles from Rat-1 cells expressing only Myc-tagged rIPK1 under the control of the tetracycline-inducible system. In the absence of doxycycline, the HPLC profile was similar to control cells (Fig. 5A). The presence of doxycycline induced IPK1 expression, and the I(1,3,4,5,6)P5 peak shifted to IP6 (Fig. 5C). Fig. 5, B and D, shows the HPLC profiles from Rat-1 cells that co-expressed GFP-tagged rIPK2 with Myc-tagged rIPK1. Because the expression of GFP-rIPK2 in these cells was not under the control of the tetracycline-inducible system, I(1,3,4,5,6)P5 was increased in the absence of doxycycline (Fig. 5B). With the doxycycline addition, we observed a substantial elevation in IP6 (Fig. 5D). Taken together, these data suggest that IPK2 and IPK1 activities are key determining steps in the production of IP5 and IP6 in Rat-1 cells.



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  		FIG. 5.
Inositol polyphosphate profiles of Rat-1 cells simultaneously expressing rat IPK2 and rat IPK1. Rat-1 cells stably expressing pRevTRE/myc-rIPK1 (A and C) or pRevTRE/myc-rIPK1 and pBabePuro/GFP-rIPK2 (B and D) were labeled in Medium-199 supplemented with 10% dialyzed FBS and containing 20 µCi/ml [3H]inositol for 2 days with (C and D) or without (A and B) 2 µg/ml doxycycline for 2 days. The soluble inositol polyphosphates were extracted from equal cell numbers and then analyzed by HPLC using the Partisphere strong anion exchange column as described under "Experimental Procedures." The elution positions of inositol polyphosphates were compared with the elution of known species and are indicated by the arrows.

 
Down-regulation of Rat IPK2 Using Vector-based siRNA— The data presented so far are from overexpression of genes, that is to say the effects of the gain of function. Another way of analyzing the function of gene products is by eliminating their expression to see the effects of the loss of function. To do this, we applied RNAi (32, 36, 37). To avoid experiment-to-experiment variability due to different transfection efficiencies with the in vitro synthesized siRNAs, we used a vector-based siRNA expression system (32) to knock down the rIPK2 expression. Fig. 6A shows the Northern blotting data using the entire rIPK2 open reading frame as a probe. Cells expressing siRNA against rIPK2 had a 90% decrease of rIPK2 mRNA compared with cells expressing the vector control (Fig. 6A). Control actin amounts were not changed. To determine whether the knock-down of rIPK2 effected inositol polyphosphate production, we labeled these cells using [3H]inositol, and the soluble inositol polyphosphates were isolated and analyzed by HPLC. The cells expressing siRNA against rIPK2 had decreased I(1,3,4,5,6)P5 levels to about 20–25% of the control cells (Fig. 6, B and C). The rIPK2 siRNA did not effect IP6 levels.



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  		FIG. 6.
Northern blotting and inositol polyphosphate profiles of Rat-1 cells expressing vector-based siRNA against rat IPK2. A, Northern blotting analysis of the Rat-1 cells expressing the vector control (left) or vector-based rIPK2 siRNA (right). 20 µg of total RNA was separated using a formaldehyde gel, transferred to Nylon membrane, and then hybridized with rIPK2 (top) or {beta}-actin (bottom) probe. B and C, inositol polyphosphate profiles of Rat-1 cells expressing the vector control (B) or vector-based rIPK2 siRNA (C). The labeling, extraction, and analysis were done as described under "Experimental Procedures." The elution positions of inositol polyphosphates were compared with the elution of known species and are indicated by the arrows. Ins, inositol.

 
Overexpression of IPK2 or IPK1 Activities Is Not Sufficient to Induce Anchorage-independent Growth of Rat-1 Cells—Several studies have indicated that transformation of Rat-1 cells with the oncogene v-Src results in activation of inositol trisphosphate kinase activity and elevation of IP4 levels (38–40). We therefore tested whether or not gain of function of either IPK2 or IPK1 activities in Rat-1 cells enabled anchorage-independent growth (Fig. 7). Overexpression of either scIpk2 or rIPK2 increased IP4 and IP5 levels in cells; however, soft agar analysis demonstrated that these modified cells were not able to grow (Fig. 7, B and C). In contrast v-Src-transformed Rat-1 cells were able to grow in soft agar (Fig. 7F). We next examined Rat-1 cells stably overexpressing either scIpk1 or rIPK1 in soft agar assays (Fig. 7, D and E). Despite the elevated levels of IP6 neither cell line was able to divide in an anchorage-independent manner.



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  		FIG. 7.
Anchorage-independent growth of Rat-1 cells expressing inositol polyphosphate kinase(s) in soft agar. Approximately 5 x 104 Rat-1 cells stably expressing pRevTRE/GFP (A), pRevTRE/GFP-ScIPK2 (B), pRevTRE/GFP-rIPK2 (C), pRevTRE/GFP-ScIPK1 (D), pRevTRE/GFP-rIPK1 (E), or v-Src (F) were seeded into a 35-mm culture dish containing 1 ml of culture media with 0.4% agar. The dishes had been coated with 2 ml of culture media containing 0.7% agar. Colony formation was scored after 3 weeks at 37 °C in a humidified atmosphere of 5% CO2.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
There are several reports that have described functions for the highly phosphorylated inositol polyphosphates such as IP5 and IP6 in eukaryotic cells (1–3). However, the molecular route of their synthesis has remained unclear in higher eukaryotic cells. To elucidate the molecular basis for synthesis of these important messengers we established cell lines in which inositol polyphosphate levels were altered through overexpression or RNAi of inositol polyphosphate kinases. We found that rat IPK2 and rat IPK1 play a critical role in the synthesis of IP5 and IP6. Our data suggest that rIPK2 and rIPK1 activities are rate-determining steps in rat IP5 and IP6 production and appear highly regulated. Importantly we demonstrated, both in vitro and in vivo, that rIPK2 and rIPK1 activities are sufficient to convert I(1,4,5)P3 to IP6 and thus are the minimal two kinases required for IP6 synthesis in rat cells. This observation is consistent with what we have reported in budding yeast, plants, and flies.

Our data indicate that absolute levels of IP6 in Rat-1 cells are tightly controlled such that it appears that feedback mechanisms sense and control its levels. The simultaneous overexpression of rIPK2 and rIPK1 where the level of IP6 appears to reach an upper threshold at which point it may be degraded support this hypothesis. For instance, the IP6 levels in the IPK2- and IPK1-overexpressing cells did not proportionally elevate the levels of IP6 compared with IPK1-overexpressing cells. In addition, decreasing cellular I(1,3,4,5,6)P5 levels using siRNA against rIPK2 significantly altered the ratio of IP5 to IP6. This indicates that rIPK1 activity has been up-regulated to compensate for IP6 loss.

The substrate specificity of mammalian orthologs of IPK2 is variable according to recent reports (10–12). Our data showed that rIPK2 phosphorylates I(1,4,5)P3 to I(1,3,4,5,6)P5 through I(1,3,4,5)P4 in vitro. Chang et al. (12) have reported that the human IPK2 ortholog acts primarily as a I(1,3,4,6)P4 5-kinase using kinetic studies and yeast complementation assays, which showed that the human ortholog did not complement inositol polyphosphate production in ipk2{Delta} yeast. We found that rIPK2 was able to complement IP5 and IP6 production in ipk2{Delta} yeast demonstrating its ability to convert I(1,4,5)P3 to I(1,3,4,5,6)P5. The different specificities may arise from slight sequence variation as rIPK2 is 83.6% identical to human IPK2 (12). Reports of two other groups have indicated that human IPK2 is capable of converting I(1,4,5)P3, although these studies only examined activity in vitro and did not provide proof in cells (10, 11). It is also noteworthy that the IP4 intermediate of I(1,4,5)P3 to I(1,3,4,5,6)P5 phosphorylation by rIPK2 was different from that of budding yeast, plant, and fly Ipk2, all of which prefer to first phosphorylate the D-6 position of I(1,4,5)P3 and then to phosphorylate the D-3 position to produce I(1,3,4,5,6)P5 (6, 7, 29, 41). Moreover the rIPK2 D127A mutant, which corresponds to the Scipk2 kin– (D131A) mutant (6), still showed weak but significant activity toward I(1,4,5)P3 both in vitro and in vivo. Thus it appears that subtle changes in amino acids in the active site allow for significant alterations in substrate recognition between rat and yeast Ipk2. This feature will likely be highly useful for future studies aimed at designing substrate-selective Ipk2 mutants.

Are there multiple pathways in place that enable IP6 synthesis in mammalian cells? Given the importance of these IP regulators, it is likely that alternate pathways may exist. Of interest, among the pathways proposed (see Fig. 8) the last two steps require IPK2 and IPK1 activities. In mammalian cells, one proposed alternate route involves two additional kinases and one phosphatase: 1) an IP3 3-kinase (which based on sequence similarity is a more recently evolved member of the IPK2 superfamily); 2) an I(1,3,4)P3 5/6-kinase, which generates either I(1,3,4,5)P4 or I(1,3,4,6)P4; and 3) an I(1,3,4,5)P4 5-phosphatase. Whether or not this route plays a role in IP5 and IP6 synthesis in Rat-1 cells is not known. Future studies of the rat I(1,3,4)P3 5/6-kinase will be important for determining this in Rat-1 cells. Although we clearly demonstrated in this study that rIPK2 is capable of bypassing these alternate three steps and directly phosphorylates I(1,4,5)P3 to I(1,3,4,5,6)P5 via I(1,3,4,5)P4 in yeast and Rat-1 cells. However, since our RNAi studies indicated that loss of rIPK2 diminishes but does not eliminate IP production, it is possible that the alternate pathway is contributing in part to IP6 production. It is also possible since the RNAi only partially knocked down rIPK2 that this accounts for the residual IP6 synthesis. It is also tantalizing to speculate that the other reported roles of the I(1,3,4)P4 5/6-kinase, as a regulator of protein phosphorylation (42, 43) and I(3,4,5,6)P5 metabolism (44), may function to control IP metabolism independently of I(1,3,4)P3 phosphorylation. Given that rIPK2 functions as an I(1,4,5)P3 3-kinase in vivo, it remains an important question to determine the functional redundancy or difference between IPK2 and IP3 3-kinases, which are only capable of phosphorylating the D-3 position of I(1,4,5)P3 (2). Do these two enzymes compete with each other for substrate, or are they compartmentalized to access different pools of substrate in the cells? Three molecular studies indicate that IP3 3-kinases are not involved in IP5 and IP6 synthesis. 1) Balla et al. (45) suggested that overexpression of IP3 3-kinase did not increase higher inositol polyphosphates such as IP5 and IP6 in NIH 3T3 cells; 2) IP3 3-kinase mouse knock-out cells reveal that IP5 and IP6 production is unaltered (46–48); and 3) in Drosophila melanogaster loss of IP3 3-kinase isoforms does not decrease IP6 levels, but loss of dmIpk2 nearly ablates IP5 and IP6 (41).



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  		FIG. 8.
Molecular basis for inositol polyphosphate synthesis in Rat-1 cells. Phospholipase C activation results in the conversion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to I(1,4,5)P3. In S. cerevisiae, Arabidopsis, and D. melanogaster, I(1,4,5)P3 is phosphorylated predominately first on the D-6 position to generate I(1,4,5,6)P4 and then the D-3 position to generate I(1,3,4,5,6)P5 by Ipk2 (designated ScIpk2 for the original characterization in S. cerevisiae). Our studies of rat IPK2 indicate that it prefers to convert I(1,4,5)P3 to I(1,3,4,5,6)P5 via an I(1,3,4,5)P4 intermediate. IP3 3-kinase, a recently evolved relative IPK2, also generates I(1,3,4,5)P4 as its sole product. However, given the data presented in this report, previous published work in D. melanogaster (41), and mouse knock-out data of others (46–48), it does not appear that IP3 3-kinase (IP3 3-K) contributes to the direct synthesis of IP5 and IP6 in flies, rat, or mouse cells. Similar to yeast, plant, and flies, the rat IPK1 acts as a 2-kinase to convert I(1,3,4,5,6)P5 to IP6. Finally our work does not exclude the involvement of I(1,3,4,5)P4 5-phosphatase (5-ptase) and I(1,3,4)P3 5/6-kinase (IP3 5/6-K) activities in IP6 production in mammals; however, it does demonstrate that rIPK2 and rIPK1 activities are sufficient to convert I(1,4,5)P3 to IP6 in cells.

 
Several reports have suggested the involvement of highly phosphorylated inositol polyphosphate production in cell proliferation and cell transformation (38, 49–51). We could not detect a significant difference of proliferation rate and colony formation ability among control and our inositol polyphosphate kinase overexpression and IPK2 knock-down Rat-1 cells (not shown). Thus it appears that 2.5-fold elevation or 80% reduction of IP5 does not sufficiently alter growth. It is likely that the remaining 20% activity in the RNAi knock-down cells is sufficient for survival; thus the examination of IPK2 or IPK1 complete knock-outs will be important future studies. Additionally investigating the relationship between the production of highly phosphorylated inositol polyphosphates and IPK2 and/or IPK1 activity during the cell cycle, cell differentiation, proliferation, and transformation may be equally important.

In conclusion, in this study we established an in vivo model system to modulate the inositol polyphosphate levels. Importantly we found that, in Rat-1 cells, rIPK2 and rIPK1 are the minimal kinase activities required to synthesize IP6 from I(1,4,5)P3 via I(1,3,4,5)P4 and I(1,3,4,5,6)P5 intermediates. This now confirms that in S. cerevisiae (5, 6), Arabidopsis (29), D. melanogaster (41), and now Rattus norvegicus these two kinases are functionally important. The ability to modulate the intracellular inositol polyphosphate levels will provide powerful tools to study the roles of I(1,3,4,5,6)P5 and IP6 in eukaryotic cell signaling.


   FOOTNOTES
 
* This work was supported by funds from the Howard Hughes Medical Institute and by National Institutes of Health R01 Grant HL-55672. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY823319 [GenBank] . Back

{ddagger} To whom correspondence should be addressed: Dept. of Pharmacology and Cancer Biology, Howard Hughes Medical Inst., 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; rIPK2, rat inositol polyphosphate kinase 2; rIPK1, rat inositol polyphosphate kinase 1; HPLC, high pressure liquid chromatography; IP, inositol polyphosphate; I(1,3,4)P3, inositol 1,3,4-trisphosphate; I(1,4,5,6)P4, inositol 1,4,5,6-tetrakisphosphate; I(1,3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate; I(1,3,4,6)P4, inositol 1,3,4,6-tetrakisphosphate; I(1,3,4,5,6)P5, inositol 1,3,4,5,6-pentakisphosphate; IP3, inositol trisphosphate; IP4, inositol tetrakisphosphate; IP5, inositol pentakisphosphate; IP6, inositol hexakisphosphate; RNAi, RNA interference; siRNA, short interference RNA; FBS, fetal bovine serum; RT, reverse transcription; GFP, green fluorescent protein; GST, glutathione S-transferase; At, A. thaliana; Sc, S. cerevisiae; dm, D. melanogaster; I(1,4)P2, inositol 1,4-bisphosphate; I(1)P, inositol 1-phosphate. Back

2 J. Stevenson-Paulik, R. J. Bastidas, S. T. Chiou, R. A. Frye, and J. D. York, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Tso-Pang Yao (Duke University Medical Center) for kindly providing the retrovirus expression vector, pBabePuro, and phoenix cell line. We also thank Dr. Ann Marie Pendergast (Duke University Medical Center) for kindly providing the v-Src-expressing Rat-1 cells. We thank Dr. Jill Stevenson-Paulik for critical discussion and reading the manuscript and June dela Cruz for technical support.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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