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J. Biol. Chem., Vol. 277, Issue 46, 43836-43843, November 15, 2002

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The Human Homolog of the Rat Inositol Phosphate Multikinase Is an Inositol 1,3,4,6-Tetrakisphosphate 5-Kinase^*

Shao-Chun Chang, Aimee L. Miller^§, Yucheng Feng, Susan R. Wente^§, and Philip W. Majerus^¶

From the  Department of Internal Medicine, Division of Hematology and the ^§ Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, June 20, 2002, and in revised form, September 9, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have demonstrated that the human homolog of the rat inositol phosphate multikinase is an inositol 1,3,4,6-tetrakisphosphate 5-kinase (InsP₄ 5-kinase). The cDNA of the human gene contained a putative open reading frame of 1251 bp encoding 416 amino acids with 83.6% identity compared with the rat protein. The substrate specificity of the recombinant human protein demonstrated preference for Ins(1,3,4,6)P₄ with a catalytic efficiency (V_max/K_m) 43-fold greater than that of Ins(1,3,4,5)P₄ and 2-fold greater than that of Ins(1,4,5)P₃. The apparent V_max was 114 nmol of Ins(1,3,4,5,6)P₅ formed/min/mg of protein, and the apparent K_m was 0.3 µM Ins(1,3,4,6)P₄. The functional homolog in yeast is Ipk2p, and ipk2-null yeast strains do not synthesize Ins(1,3,4,5,6)P₅ or InsP₆. Synthesis of these compounds was restored by transformation with wild-type yeast IPK2 but not with human InsP₄ 5-kinase. Thus the human gene does not complement for the loss of the yeast gene because yeast cells do not contain the substrate Ins(1,3,4,6)P₄, and the reaction of the human protein with Ins(1,3,4,5)P₄ is insufficient to effect rescue or synthesis of InsP₅ and InsP₆. Therefore the major activity of human InsP₄ 5-kinase is phosphorylation at the D-5 position, and the pathways for synthesis of Ins(1,3,4,5,6)P₅ in yeast versus humans are different.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inositol phosphates are a group of highly complex molecules for which cellular functions are being defined; however, this process has been difficult because of the large number of different inositol phosphates, with varying numbers of phosphates in different positions of the inositol ring. The synthetic pathway generating higher order inositol phosphates, inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P₅)¹ and inositol hexakisphosphate (InsP₆), in human cells is not completely defined. Current evidence supports inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) as the common precursor and Ins(1,3,4,5,6)P₅ and InsP₆ as the predominant higher inositol phosphates in human cells, and the synthetic pathway from Ins(1,4,5)P₃ to Ins(1,3,4,5,6)P₅ remains an area of active investigation (for general reviews, see Refs. 1 and 2). Hence, the goal of the current study is to define a catalytic step leading to Ins(1,3,4,5,6)P₅ synthesis.

The best characterized InsP₆ synthetic pathway is in Saccharomyces cerevisiae (3). The common precursor Ins(1,4,5)P₃ is synthesized from phospholipase C and subsequently used by two distinct proteins, Ipk2p and Ipk1p, ultimately to generate InsP₆. The IPK1 and IPK2 genes were isolated from a genetic screen for mutants that were synthetically lethal in combination with a gle1-2 mutant, which is defective in mRNA export. Sequential phosphorylation of Ins(1,4,5)P₃ at the D-6 and D-3 positions generates Ins(1,4,5,6)P₄ and Ins(1,3,4,5,6)P₅, respectively, catalyzed by the IPK2 gene product (4). Next, phosphorylation at the D-2 position yields InsP₆ catalyzed by the IPK1 gene product (5).

In human cells, the synthetic pathway to higher inositol phosphates shares the same initial step to produce the common precursor Ins(1,4,5)P₃ catalyzed by phospholipase C (2). However, the pathway from Ins(1,4,5)P₃ to Ins(1,3,4,5,6)P₅ is less clear. Two different synthetic pathways generating different isomers of InsP₄ have been described. First, the synthesis of Ins(1,3,4,5)P₄ from Ins(1,4,5)P₃ has been well documented to be catalyzed by inositol 1,4,5-trisphosphate 3-kinases (InsP₃ 3-kinases) in response to stimulation by various ligands (6, 7). Genes encoding the InsP₃ 3-kinase have been cloned (8-10) and the enzymatic catalysis characterized (6). Second, the synthesis of Ins(1,3,4,6)P₄ from Ins(1,3,4)P₃ was described to be catalyzed by the inositol 1,3,4-trisphosphate 5/6-kinase (InsP₃ 5/6-kinase), which has no activity toward the Ins(1,4,5)P₃ isomer (11). The Ins(1,3,4)P₃ isomer is synthesized from Ins(1,3,4,5)P₄ catalyzed by 5-phosphatase(s). The human enzymes responsible for the conversion of either Ins(1,3,4,5)P₄ or Ins(1,3,4,6)P₄ to Ins(1,3,4,5,6)P₅ have not been reported. The final step is the phosphorylation of Ins(1,3,4,5,6)P₅ at the D-2 position catalyzed by inositol 1,3,4,5,6-pentakisphosphate 2-kinase (12).

To date, the synthesis of Ins(1,3,4,5,6)P₅ from InsP₄ has been characterized in S. cerevisiae, as noted above, and in rat. The rat inositol phosphate multikinase (IPMK) was isolated based on sequence homology at the putative binding site that catalyzed the transfer of phosphate from ATP to inositol phosphate (13). Saiardi and co-workers (13) present data to suggest the in vitro conversion of Ins(1,4,5)P₃ to Ins(1,3,4,5,6)P₅ via two sequential phosphorylation reactions catalyzed by the rat IPMK, first at the D-3 position followed by the D-6 position; however, definitive kinetic data and substrate specificity of the rat IPMK were not presented. The presence of a 5-kinase activity converting Ins(1,3,4,6)P₄ to Ins(1,3,4,5,6)P₅ has been previously proposed (14-16); however, the protein and the gene responsible for this reaction have not been characterized.

In this paper we report the isolation of a human inositol kinase that has homology with the rat IPMK. Analysis of substrate specificity and enzyme kinetics demonstrates a preference for phosphorylation at the D-5 position. Based on substrate preference for Ins(1,3,4,6)P₄ over Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄, we assigned the name inositol 1,3,4,6-tetrakisphosphate 5-kinase (InsP₄ 5-kinase). We postulate that human InsP₄ 5-kinase exists in a metabolic pathway downstream from the InsP₃ 5/6-kinase and upstream from InsP₅ 2-kinase. Therefore with these three human kinases, we have established a metabolic pathway from InsP₃ to InsP₆ in human cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- All chemicals were reagent grade or better. Restriction endonucleases, DNA-modifying enzymes, and general reagents were from Amersham Biosciences, Roche Molecular Biochemicals, Fisher, Invitrogen, New England Biolabs, Promega, and Stratagene unless stated otherwise. PCR was performed using the Pfu DNA polymerase per protocol from Stratagene. Oligonucleotide synthesis and DNA sequencing were performed by the Protein and Nucleic Acid Chemistry Laboratory, Washington University. Acrylamide solution, Bio-Safe Coomassie Stain, the Bradford Protein Assay Kit, Mini-Protean II Cell, and Mini Trans-Blot Cell used for protein work were purchased from Bio-Rad. The SuperSignal West Pico Kit used for detection of Western transfer blots was from Pierce. Radiolabeled inositol phosphates [³H]Ins(1,4,5)P₃, [³H]Ins(1,3,4,5)P₄, and [³H]InsP₆ were purchased from PerkinElmer Life Sciences and Amersham Biosciences. General reagents and chemicals were purchased from Sigma and Fisher unless stated otherwise.

Strains, Plasmids, and Growth Conditions-- Methods for Escherichia coli growth and selection were described previously (17, 18). E. coli strain XL-1Blue (Stratagene) was used as the bacterial host for all plasmids unless stated otherwise. Bacterial strains were cultured in LB (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.4) medium supplemented with 100 µg/ml ampicillin where appropriate and transformed by standard methods (17, 18). All bacterial strains were grown at 37 °C. Agar plates were made by the addition of 1.5% agar to the described medium. Methods for S. cerevisiae growth and selection were described previously (19-21). The S. cerevisiae strains used in this study were YOL126 (MATa; ipk2::HIS3; ura3; trp1; leu2; his3; ade2; can1) and SWY1852 (MAT; ipk2-3; gle1-2; ade2; ade3; ura3; his3; leu2; trp1; LYS2) transformed with pSW611 (GLE1/URA3/ADE3) (4). Yeast strains were grown either in 1% yeast extract and 2% peptone or in synthetic minimal medium plus appropriate amino acids supplemented with 2% glucose or 2% galactose as indicated. Yeast transformations were completed by the lithium acetate method (22). 5-Fluoroorotic acid (5-FOA, U. S. Biologicals) was used at a concentration of 1.0 mg/ml. Yeast strains were grown at 23, 30, or 37 °C as indicated.

Synthesis of Radiolabeled Inositol Phosphates-- [³H]Ins(1,3,4,6)P₄ was synthesized from [³H]Ins(1,3,4,5)P₄ (PerkinElmer Life Sciences) in a concerted reaction catalyzed by recombinant OCRL inositol polyphosphate 5-phosphatase expressed in Sf9 cells (23) and glutathione S-transferase-tagged recombinant Ins(1,3,4)P₃ 5/6-kinase expressed in E. coli (11) as follows. Excess amounts of OCRL 5-phosphatase and Ins(1,3,4)P₃ 5/6-kinase were added to 2-ml reaction mixtures containing 50 mM HEPES, pH 7.5, 3 mM MgCl₂, 250 µl of [³H]Ins(1,3,4,5)P₄ (~ 2.5 × 10⁶ cpm), 5 mM ATP, 10 mM phosphocreatine, 800 units of phosphocreatine kinase, and 1 mM dithiothreitol; the reaction was incubated at 37 °C for 1-2 h. The product was confirmed by its elution just before a [³²P]Ins(1,3,4,5,)P₄ standard on an Adsorbosphere strong anion exchange (SAX) HPLC column (Alltech) with a linear gradient of 0-100% 1 M ammonium phosphate, pH 3.5, at 1 ml/min over 120 min as published previously (11, 24). The [³²P]Ins(1,3,4,5,)P₄ standard was produced as described previously (11). The [³²P]Ins(1,3,4,5,6)P₅ standard was produced from Ins(3,4,5,6)P₄ by chick red blood cell extract (24). The [³²P]Ins(1,4,5,)P₃ was prepared as described previously (25).

Identification and Isolation of Human Ins(1,3,4,6)P₄ 5-Kinase-- The rat IPMK sequence (13) was used to screen the human EST data bank using the TBLASTN program (26). The EST clone containing human InsP₄ 5-kinase (GenBank accession number BC016612) was obtained from the American Type Culture Collection. Using the EST clone as template, the InsP₄ 5-kinase ORF was amplified by PCR using the primers 5'-CGCGGATCCGCAACAGAGCCACCATCCCCCCTCC-3' and 5'-CGGGGTACCGAGGATTCAATTGTCTAAAATACTTC-3'. The DNA sequence of both strands of the PCR product was obtained from cycle sequence PCR using ABI PRISM^® Big Dye^TM Terminators v 3.0 Cycle Sequencing Kit, and the product was analyzed at the Protein and Nucleic Acid Chemistry Laboratory at Washington University.

Site-directed Mutagenesis-- Site-directed mutagenesis was performed using QuikChange site-directed mutagenesis kit (Stratagene) as per manufacturer's protocol. Mutagenesis primers designed per manufacturer's protocol were 5'-GGATGTAAAGATAGGGCAAAAAAGCTATGATCCTTTTGC-3' and 5'-GGCAAAAGGATCATAGCTTTTTTGCCCTATCTTTACATCC-3'.

Expression and Purification of Recombinant Protein-- For expression in E. coli, a PCR product encoding human InsP₄ 5-kinase flanked by 5-prime BamHI and 3-prime KpnI sites was subcloned into the BamHI and KpnI sites of the expression vector pTrcHisA (Invitrogen). The N-terminal His-tagged fusion protein was transformed into E. coli strain BL21CodonPlus(DE3)-RIL (Stratagene). E. coli strain BL21CodonPlus(DE3)-RIL containing the expression construct (0.5 liter) was grown to O.D. ~0.6 in LB + ampicillin (100 µg/ml), and the expression was induced by adding isopropyl-1-thio--D-galactopyranoside to a final concentration of 1 mM for 4 h. Cells were harvested by centrifugation at 1,000-3,000 × g for 15 min at 4 °C, and the pellet was resuspended in 20 ml of 100 mM Tris-Cl, pH 7.5, 0.9% (w/v) NaCl, 1 mM phenylmethylsulfonyl fluoride, and 1 mM -mercaptoethanol at 4 °C. Lysozyme (Sigma) was added to final concentration of 0.75 mg/ml, and the resuspended cells were incubated at 4 °C for 60 min. Cells were lysed by sonication three times for 30 s each with a 2-min pause on ice between each sonication. Insoluble material was removed by centrifugation at 10,000-12,000 × g for 20 min at 4 °C, and the resulting cell extract was applied to 0.5 ml of cobalt-based metal affinity TALON resin (Clontech). The resin was washed three times with 10 bed volumes of 100 mM Tris-Cl, pH 7.5, 0.9% (w/v) NaCl, 1 mM phenylmethylsulfonyl fluoride, and 1 mM -mercaptoethanol at 4 °C. The His-tagged human InsP₄ 5-kinase was eluted with 100 mM imidazole in the above buffer.

For expression in S. cerevisiae, human InsP₄ 5-kinase was subcloned into the yeast-E. coli shuttle vector pLGSD5Trp under the regulation of the inducible yeast Gal10 promoter. The vector pLGSD5Trp (from John W. Verbsky) was constructed by substituting the StuI fragment containing the majority of the URA3 gene from the vector pLGSD5 with the StuI-PuvII fragment containing the TRP1 gene by blunt end ligation. The InsP₄ 5-kinase ORF was amplified by PCR using the primers 5'-CGCGGATCCATGGCAACAGAGCCACCATCC-3' and 5'-CCCGAGCTCGAGGATTCAATTGTCTAAAATAC-3'. A PCR product encoding human InsP₄ 5-kinase flanked by 5-prime BamHI and 3-prime SacI sites was subcloned into the plasmid pCR2.1-TOPO after incubation with TaqDNA polymerase per TOPO TA cloning kit protocol (Invitrogen). The BamHI-SacI fragment containing the InsP₄ 5-kinase was subcloned into pLGSD5Trp behind the yeast Gal10 promoter. The DNA sequence was verified by sequencing both strands of the PCR product as described previously.

Northern Blot Analysis-- A PCR product containing the entire ORF of human InsP₄ 5-kinase (as described above) was used as a template for random sequence hexanucleotide-mediated labeling reaction as per RediprimeII labeling kit protocol (Amersham Biosciences). The resulting ³²P-labeled probes were hybridized to Multiple Tissue Northern Blots and Multiple Tissue Expression Arrays following the manufacturer's instructions (Clontech).

Enzyme Activity Assay-- Enzymatic activity was determined by the addition of the desired amount of enzyme to 50 mM HEPES, pH 7.2, 100 mM KCl, 100 µg/ml bovine serum albumin, 8 mM MgCl₂, 5 mM ATP, and 1,000-2,000 cpm of [³H]Ins(1,3,4,6)P₄ to a total reaction volume of 50 µl at 37 °C for the desired times. The reaction was stopped by the addition of 1 ml of H₂O, and the sample was loaded onto a 500-µl Dowex AG 1-X8 formate column (mesh 200-400, Bio-Rad) equilibrated in water. The column was washed with 1 M ammonium formate and 0.1 M formic acid to elute the substrate. The product was eluted with 2 M ammonium formate and 0.1 M formic acid and counted in a liquid scintillation counter.

Product Identification-- Samples from enzymatic reactions as described above were analyzed using HPLC with either a 4.6 x 125-mm Partisphere (Whatman) SAX or a 4.6 x 250-mm Adsorbosphere SAX column as indicated. For the Partisphere SAX column, reaction products were analyzed using a linear gradient from 10 mM to 1.7 M ammonium phosphate, pH 3.5, at a flow rate of 1 ml/min, over 25 min, followed by isocratic elution with 1.7 M ammonium phosphate for 25 min. For the Adsorbosphere SAX column, reaction products were analyzed using a linear gradient of 0-1.0 M ammonium phosphate, pH 3.5, over 120 min at a flow rate of 1 ml/min, using a modification of the method of Hughes et al. (27). Radioactivity from both columns was measured using an inline detector -RAM (IN/US System Inc.), and the individual inositol polyphosphates were assigned on the basis of coelution with known samples.

In Vivo Analysis of Soluble Inositol Phosphates-- The yeast strain YOL126 harboring a null mutation in IPK2 (ipk2) was transformed with plasmids carrying human InsP₄ 5-kinase, S. cerevisiae IPK2, or an empty vector. Strains were grown to late logarithmic phase in synthetic complete medium containing 2% glucose (uninduced) or 2% galactose (induced) and 30 µCi/ml [³H]inositol. Soluble inositol phosphates were isolated as described previously (28). Samples were separated by HPLC over a Partisphere SAX column (Whatman, 4.6 × 125 mm) using a 25-min linear gradient from 10 mM to 1.7 M ammonium phosphate, pH 3.5, followed by another 25 min at 1.7 M. Peaks were identified by comparison with known inositol phosphate standards.

Functional Complementation of S. cerevisiae IPK2-- The yeast strain SWY1852 carrying synthetically lethal mutations in gle1-2 and ipk2-3 covered by pGLE1/URA3/ADE3 was transformed with plasmids carrying human InsP₄ 5-kinase or S. cerevisiae IPK2. The ability of the resulting strains to survive without pGLE1/URA3/ADE3 was tested by growth on medium containing 2% galactose and 1.0 mg/ml 5-FOA. Growth was compared with the parental strain and control strains with either a wild-type URA3 or ura3 mutation.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of Human InsP₄ 5-Kinase-- Because the yeast Ipk2p and rat IPMK proteins catalyzed the synthesis of inositol pentakisphosphate, we postulated that the human homolog might function as an InsP₄ kinase. Using the sequence of rat IPMK, a human EST (GenBank accession number BC016612) was identified. The EST contained a series of 7 adenosines at bp 446-452 compared with 6 adenosines found in the genomic sequence located on chromosome 10 (data not shown). The homology of the rat sequence extended to the entire ORF using the chromosomal sequence, suggesting that the EST sequence was in error (Fig. 1). Hence, site-directed mutagenesis was performed to produce the correct sequence of 6 adenosines. The human InsP₄ 5-kinase cDNA contains a putative ORF of 1251 bp encoding 416 amino acids (estimated 47.2 kDa); the encoded amino acids are 83.6% identical compared with the rat IPMK and 15.8% compared with the yeast Ipk2p (Fig. 1). Amino acids 140-148 (gray box in Fig. 1) contained the conserved sequence P-X-X-X-D-X-K-X-G demonstrated previously as the putative binding site that catalyzes the transfer of phosphate from ATP to inositol phosphates (29). This sequence has been demonstrated in the following inositol phosphate kinases: human Ins(1,4,5)P₃ 3-kinase (29), rat IPMK (13), mouse InsP₆K1 (30), human InsP₆K2 (30), human InsP₆K3 (31), and yeast Ipk2p (ArgRIII/Arg82) (32).

View larger version (88K): [in this window] [in a new window]   Fig. 1.   Amino acid alignment of human Ins(1,3,4,6)P4 5-kinase, yeast Ipk2p, and rat IPMK. All amino acid sequences are full-length for the indicated proteins. Regions of identity (as indicated by black boxes) were determined by using the Clustal V method (36). The conserved sequence P-X-X-X-D-X-K-X-G containing the putative active site for phosphate transfer from ATP to inositol phosphate is shown in gray.

Tissue Expression-- Northern blot analysis of different human tissues revealed two transcripts of ~6 and 4 kb which were expressed ubiquitously in all tissues tested; however, the highest expression was seen in the liver, followed by skeletal muscle, placenta, and peripheral blood leukocytes (Fig. 2). A similar result was obtained using Multiple Tissue Expression Arrays (data not shown). Hence, Northern blot analysis indicated ubiquitous endogenous expression of the identified human InsP₄ 5-kinase. Interestingly, the tissue expression of the rat IPMK is quite different with highest expression in kidney and brain (13); the significance of this difference is unclear at this time.

View larger version (71K): [in this window] [in a new window]   Fig. 2.   Tissue expression of human InsP4 5-kinase. Northern blot analysis of InsP4 5-kinase mRNA expression was performed, directed against various human tissues as indicated. Random primed 32P-labeled probe synthesized from a PCR product containing the entire ORF of the human InsP4 5-kinase was hybridized to the human 12-lane Multiple Tissue Northern Blot (Clontech). Molecular weight standards are indicated in kb on the left.

In Vitro Enzymatic Activity-- The human InsP₄ 5-kinase was subcloned into a His-tagged expression system, expressed in E. coli with isopropyl-1-thio--D-galactopyranoside induction, and purified using a cobalt-based metal affinity resin as described under "Experimental Procedures." The expression of the N-terminal His-tagged InsP₄ 5-kinase was verified by SDS-PAGE and Western blot analysis using anti-Xpress-horseradish peroxidase antibody (Invitrogen); the purified recombinant human InsP₄ 5-kinase accounted for greater than 75% of the protein in the purified fraction as determined by Coomassie Blue staining (data not shown). The purified InsP₄ 5-kinase phosphorylated Ins(1,3,4,6)P₄ to produce Ins(1,3,4,5,6)P₅, whereas proteins isolated from bacterial cells containing vector alone did not exhibit any inositol phosphorylation activity (data not shown). This result established that the inositol kinase activity was from the His-tagged human InsP₄ 5-kinase and not from the copurified E. coli proteins.

Substrates of the Human InsP₄ 5-Kinase-- Previous reports of the yeast Ipk2p and rat IPMK predicted multiple substrate specificity and catalysis of sequential phosphorylations to yield Ins(1,3,4,5,6)P₅ (4, 13). Hence, the substrate specificity of the human homolog was evaluated. Either 5 or 40 ng of human InsP₄ 5-kinase was incubated with 200 nM Ins(1,3,4,6)P₄, 100 nM Ins(1,3,4,5)P₄, and 100 nM Ins(1,4,5)P₃ as substrates for 5 min at 37 °C (for details, see Fig. 3). The InsP₄ 5-kinase was able to synthesize InsP₅ from both isomers of InsP₄ (Fig. 3, B and C) and InsP₄ from Ins(1,4,5)P₃ (Fig. 3D). Interestingly, the Ins(1,3,4,6)P₄ was the best substrate compared with Ins(1,3,4,5)P₄ and Ins(1,4,5)P₃ (note 8-fold less enzyme used in 3B compared with 3C and 3D), and the Ins(1,4,5)P₃ reaction did not produce any InsP₅ or PP-InsP₄ (Fig. 3D). The small peak at the InsP₅ elution position was not significantly above the background. Ins(1,3,4)P₃ was not a substrate (data not shown). The human InsP₄ 5-kinase exhibited a substrate preference for Ins(1,3,4,6)P₄.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   HPLC of InsP4 5-kinase reactions. The human InsP4 5-kinase reaction was incubated for 5 min at 37 °C with different substrates, and the reaction products were identified using a Partisphere SAX HPLC column as described under "Experimental Procedures." A shows three reactions without the addition of enzyme (i.e. substrates). The right y axis is for Ins(1,3,4,6)P4 (open circles) and Ins(1,3,4,5)P4 (open squares), and the left y axis is for Ins(1,4,5)P3 (closed circles). Subsequent panels are for reactions with each individual substrate as follows: B, 5 ng of InsP4 5-kinase and 200 nM [3H]Ins(1,3,4,6)P4 (open circles); C, 40 ng of InsP4 5-kinase and 100 nM [3H]Ins(1,3,4,5)P4 (open squares); and D, 40 ng of InsP4 5-kinase and 100 nM [3H]Ins(1,4,5)P3 (closed circles). Peaks were identified by comparison with known inositol phosphate standards.

Product Identification-- To identify the products of InsP₄ 5-kinase definitively, an Adsorbosphere SAX HPLC column was used which can resolve different isomers of the higher phosphorylated inositol phosphates. The human InsP₄ 5-kinase catalyzed the conversion of Ins(1,3,4,6)P₄ to Ins(1,3,4,5,6)P₅, as shown by coelution with standard [³²P]Ins(1,3,4,5,6)P₅ on HPLC (Fig. 4). Our initial experiment using Ins(1,4,5)P₃ as substrate synthesized no detectable amounts of InsP₅ or PP-InsP₄; hence, the InsP₄ 5-kinase was incubated with the substrate Ins(1,4,5)P₃ as function of time. The production of Ins(1,3,4,5)P₄ was identified by coelution with the [³²P]Ins(1,3,4,5,)P₄ standard (Fig. 5, B-D); additionally, the InsP₄ product was dephosphorylated by the 5-phosphatase OCRL (33) (Fig. 5E). This further supported Ins(1,3,4,5,)P₄ as the product and eliminated Ins(1,4,5,6)P₄ as a product because Ins(1,4,5,6)P₄ would be resistant to dephosphorylation by 5-phosphatase. Interestingly, neither the production of InsP₅ nor that of PP-InsP₄ was observed (Fig. 5D). Hence, human InsP₄ 5-kinase was shown to have 5-kinase and 3-kinase activity. No 6-kinase activity was seen using our experimental conditions.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Identification of the InsP4 5-kinase reaction product with Ins(1,3,4,6)P4 as substrate. Human InsP4 5-kinase (1 ng) was incubated with [3H]Ins(1,3,4,6)P4 for 0 min (A) and 10 min (B), and the product of the reaction (left y axis) was analyzed using an Adsorbosphere SAX HPLC column as described under "Experimental Procedures." [32P]Inositol polyphosphate standards (right y axis) were added after termination of the reaction and prior to injection of the sample onto HPLC.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Identification of the InsP4 5-kinase reaction product with Ins(1,4,5)P3 as substrate. Human InsP4 5-kinase (1 ng) was incubated with 24 µM [32P]Ins(1,4,5)P3 for 0 min (A), 30 min (B), 120 min (C), and 360 min (D). The 360-min reaction was treated with OCRL 5-phosphotase (E). Products of the reaction (right y axis) were analyzed using an Adsorbosphere SAX HPLC column as described under "Experimental Procedures." [3H]Inositol phosphate standards (left y axis) were added after termination of the reaction and prior to injection.

Kinetic Analysis-- To determine the substrate preference of the human InsP₄ 5-kinase Michaelis-Menten kinetics were performed using Ins(1,3,4,6)P₄, Ins(1,3,4,5)P₄, and Ins(1,4,5)P₃ as substrates. The conversion of substrate to product was determined at a fixed amount of enzyme and a given substrate concentration at time 0, 10, 20, and 30 min; the resulting product versus time plot generated a line using the least mean squares method whose slope represented the velocity of the reaction at the given substrate concentration (data not shown). The conversion of substrate to product was linear with respect to time within our experimental system, indicating that product inhibition is unlikely under our experimental conditions. Next the velocity was plotted against substrate concentration, and the kinetic parameters K_m and V_max were determined from nonlinear curve fit of the Michaelis-Menten equation (Fig. 6). The K_m and V_max for each substrate were as follows: for Ins(1,3,4,6)P₄, K_m = 295 nM and V_max = 114 nmol of InsP₅ formed/min/mg; for Ins(1,3,4,5)P₄, K_m = 129 nM and V_max = 1.1; and for Ins(1,4,5)P₃, K_m = 112 nM and V_max = 27. The above kinetic values translate to catalytic processivities (k_cat), derived from V_max/K_m, of 0.39 liter/min/mg for Ins(1,3,4,6)P₄, 0.009 liter/min/mg for Ins(1,3,4,5)P₄, and 0.24 liter/min/mg for Ins(1,4,5)P₃. Kinetically, Ins(1,3,4,6)P₄ is 43 times better as a substrate compared with Ins(1,3,4,5)P₄ and 1.6 times better substrate compared with Ins(1,4,5)P₃. Hence, human InsP₄ 5-kinase specificity for the different substrates would be in the following order: Ins(1,3,4,6)P₄ > Ins(1,4,5)P₃ Ins(1,3,4,5)P₄.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Kinetics of human Ins(1,3,4,6)P4 5-kinase. Recombinant His-tagged human Ins(1,3,4,6)P4 5-kinase purified from E. coli was assayed for enzymatic activity as described under "Experimental Procedures." The ATP concentration was fixed at 5 mM while varying the substrate concentration of Ins(1,3,4,6)P4 (A), Ins(1,3,4,5)P4 (B), and Ins(1,4,5)P3 (C). A nonlinear curve fit to the Michaelis-Menten equation using the least squares method was performed using KaleidaGraph version 3.5.1 (Synergy Software); Km, Vmax, and R values for each curve are listed. The activity was normalized to protein. The plot shown is representative of multiple experiments.

In Vivo Analysis of the Human InsP₄ 5-Kinase-- The ability of human InsP₄ 5-kinase to act on Ins(1,4,5)P₃ in vivo was tested using S. cerevisiae harboring a null mutation in IPK2 (ipk2), also known as ARG82 or ARGRIII. Because Ipk2p sequentially phosphorylates Ins(1,4,5)P₃ to produce Ins(1,4,5,6)P₄ and Ins(1,3,4,5,6)P₅, the ipk2 yeast accumulates Ins(1,4,5)P₃ (Fig. 7C) (4). Thus, the ipk2 genetic background provides an in vivo pool of Ins(1,4,5)P₃ ideal for evaluating the activity of human InsP₄ 5-kinase. Fig. 7 shows the HPLC analysis of soluble inositol phosphates isolated from yeast grown in the presence of [³H]inositol (28). The metabolic pathway from Ins(1,4,5)P₃ to InsP₆ was restored by introduction of a plasmid-borne copy of S. cerevisiae IPK2 (Fig. 7D). The inositol phosphate profile for the ipk2 strain expressing human InsP₄ 5-kinase demonstrated that only a small amount of Ins(1,4,5)P₃ was converted to InsP₄ and that the InsP₄ made was not converted to InsP₅ and InsP₆ as observed when the yeast Ipk2p enzyme was present (Fig. 7, D versus E). Ins(1,3,4,5)P₄ is the likely product given the above in vitro data and the fact that the InsP₄ peak disappeared after OCRL 5-phosphatase incubation (data not shown). There were two peaks, one just after InsP₄ and the second just before InsP₅, which remained unidentified and may represent different isomers. These results indicated that human InsP₄ 5-kinase does not form Ins(1,3,4,5,6)P₅ in the yeast ipk2 strain and cannot function as part of the InsP₆ biosynthetic pathway defined for yeast (34).

View larger version (35K): [in this window] [in a new window]   Fig. 7.   Functional complementation of yeast ipk2 mutants. HPLC analysis of soluble inositol phosphates isolated from yeast strains grown in the presence of 30 µCi/ml [3H]inositol was performed as described under "Experimental Procedures." Inositol phosphate standards are noted by arrows above the chromatograms. HPLC chromatograms are shown for wild-type yeast (A), ipk1 strain possessing a wild-type copy of IPK2 but lacking the 2-kinase for InsP6 synthesis (B), ipk2-3 strain grown at 23 °C, which can synthesize Ins(1,4,5,6)P4 (C), ipk2 strain transformed with plasmid bearing the yeast IPK2 (D), or human InsP4 5-kinase under induced condition (2% galactose) (E), and ipk2-3 strain with plasmid bearing the human InsP4 5-kinase under induced condition (2% galactose) (F).

To address whether functional complementation can be achieved in a partially active IPK2 background, the ipk2-3 mutant yeast was transformed with human InsP₄ 5-kinase. The ipk2-3 allele encodes a mutant Ipk2p that is able to synthesize Ins(1,4,5,6)P₄ but not InsP₅ at 23 °C (Fig. 7B) (34). If human InsP₄ 5-kinase can utilize this isomer of InsP₄, the presence of InsP₆ would be detected given the presence of a functional Ipk1p. The HPLC analysis of [³H]inositol-labeled ipk2-3 cells showed that human InsP₄ 5-kinase was able to phosphorylate Ins(1,4,5,6)P₄ synthesizing InsP₅ (Fig. 7F); this was also observed for wild-type Ipk2p (data not shown). Additionally, the ability of human InsP₄ 5-kinase to rescue a genetic synthetic lethal phenotype between yeast IPK2 and GLE1, which encodes an mRNA export factor (4, 34), was examined. The combination of mutations in both of these genes renders the yeast strain inviable unless a wild-type copy of one of the genes is present on a plasmid. The presence of the rescuing plasmid can be followed by the inclusion of the URA3 gene on the same plasmid. Only strains that have lost the plasmid carrying the wild-type URA3 gene are able to grow in the presence of 5-FOA (35). Because human InsP₄ 5-kinase can utilize Ins(1,4,5,6)P₄ as noted above, the ipk2-3/gle1-2 mutant yeast would no longer require the presence of the rescuing plasmid and would lose the URA3 gene; therefore, the cells would be able to grow in the presence of 5-FOA. As seen in Fig. 8, the yeast strain with ipk2-3 and gle1-2 mutations was viable on 5-FOA medium in the presence of either wild-type Ipk2p (Fig. 8A) or human InsP₄ 5-kinase (Fig. 8E). Hence, human InsP₄ 5-kinase can only complement the yeast IPK2 defect when partial function of the Ipk2p exists and Ins(1,4,5,6)P₄ is provided as a substrate.

View larger version (60K): [in this window] [in a new window]   Fig. 8.   Analysis of the rescue of the synthetic lethal phenotype in ipk2-3/gle1-2 mutant. Yeast strains were grown on medium containing 2% galactose and 5-FOA. Strains are as follows: A, ipk2-3/gle1-2 + pIPK2/LEU2; B, ipk2-3/gle1-2 + pGLE1/URA3/ADE3; C, ura3 control; D, URA3 control; E, ipk2-3/gle1-2 + phumanInsP4 5-kinase/GAL1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have elucidated a pathway leading to the synthesis of InsP₆ in human cells. This pathway starts with Ins(1,4,5)P₃ and ends at InsP₆. The final step in the synthesis of InsP₆ from Ins(1,3,4,5,6)P₅ is catalyzed by a 2-kinase that was characterized recently (12). However, the pathway from Ins(1,4,5)P₃ to Ins(1,3,4,5,6)P₅ has not been fully defined. In this paper, we report results supporting the synthesis of Ins(1,3,4,5,6)P₅ from Ins(1,3,4,6)P₄ catalyzed by Ins(1,3,4,6)P₄ 5-kinase.

In previous work, the synthesis of Ins(1,3,4,5,6)P₅ has been demonstrated by two enzymes, yeast Ipk2p and rat IPMK (4, 13). Hence, we postulated that the human homolog would catalyze the synthesis of Ins(1,3,4,5,6)P₅. Using the rat IPMK amino acid sequence to search the GenBank data base, we found a human EST encoding a 416-amino acid protein with 83.6% identity to the rat IPMK and 15.8% compared with the yeast Ipk2p. The tissue expression profile of the mRNA is ubiquitous with highest expression in liver. The tissue distribution is quite different compared with rat IPMK, which has the highest expression in kidney and brain (13).

Both yeast Ipk2p and rat IPMK have been shown to have multikinase activity, i.e. they phosphorylate inositol phosphate at multiple sites. Yeast Ipk2p first phosphorylates Ins(1,4,5)P₃ at the D-6 position generating Ins(1,4,5,6)P₄ and then phosphorylates Ins(1,4,5,6)P₄ at the D-3 position generating Ins(1,3,4,5,6)P₅; these two phosphorylations are thought to occur in a concerted reaction (4). Similarly, rat IPMK has been described first to phosphorylate Ins(1,4,5)P₃ at the D-3 position, generating Ins(1,3,4,5)P₄, and then Ins(1,3,4,5)P₄ at the D-6 position, generating Ins(1,3,4,5,6)P₅ (13). The hypothesis is that Ins(1,3,4,5,6)P₅ is synthesized from Ins(1,4,5)P₃ by two sequential phosphorylation reactions catalyzed by a multikinase.

Our data support the idea that human InsP₄ 5-kinase functions primarily as a 5-kinase. In vitro analysis demonstrated the ability to utilize multiple substrates including Ins(1,3,4,6)P₄, Ins(1,3,4,5)P₄, and Ins(1,4,5)P₃; however, kinetic analysis indicates that Ins(1,3,4,6)P₄ is the preferred substrate, 40-fold and 1.6-fold over Ins(1,3,4,5)P₄ and Ins(1,4,5)P₃, respectively. Hence, the 5-kinase reaction of human InsP₄ 5-kinase has the highest catalytic efficiency.

The 3-kinase reaction of human InsP₄ 5-kinase leading to the synthesis of Ins(1,3,4,5)P₄ is robust in vitro; however, the physiological significance of this reaction is not clear. The synthesis of Ins(1,3,4,5)P₄ from Ins(1,4,5)P₃ has been well characterized to be catalyzed by the Ins(1,4,5)P₃ 3-kinases. The Ins(1,4,5)P₃ 3-kinases have significantly higher V_max (ranging from 283 nmol/min/mg for human platelets (37) to 9.5 µmol/min/mg for rat brain (38)) in the presence of calcium and are likely to be the main source of Ins(1,3,4,5)P₄ production. Additionally, human InsP₄ 5-kinase was unable to utilize the in vivo pool of Ins(1,4,5)P₃ in the ipk2 mutant yeast to synthesize a significant amount of Ins(1,3,4,5)P₄ efficiently (Fig. 7); only a small amount of Ins(1,3,4,5)P₄ was made.

The 6-kinase reaction of human InsP₄ 5-kinase leading to the synthesis of Ins(1,3,4,5,6)P₅ from Ins(1,3,4,5)P₄ is the least catalytically efficient of the reactions examined. From kinetic analysis, Ins(1,3,4,5)P₄ is not a good substrate for human InsP₄ 5-kinase. In the presence of either Ins(1,3,4,6)P₄ or Ins(1,4,5)P₃, Ins(1,3,4,5)P₄ would be competitively inhibited by the two former substrates. This was demonstrated by Fig. 5, where InsP₄ 5-kinase was incubated with Ins(1,4,5)P₃ for up to 360 min, and the production of InsP₅ was not observed. Additionally, human InsP₄ 5-kinase was unable to use Ins(1,3,4,5)P₄ in the presence of Ins(1,4,5)P₃ to produce InsP₅ in the ipk2 mutant yeast. However, in a partially active mutant of Ipk2p, human InsP₄ 5-kinase was able to synthesize InsP₅ as demonstrated by the production of InsP₆ catalyzed by Ipk1p (Fig. 7); this supports the theory that the human enzyme can interact within the yeast inositol phosphate metabolic pathway in the presence of partial Ipk2p activity.

Based on the above argument, human InsP₄ 5-kinase functions as a 5-kinase. Our data indicate that human InsP₄ 5-kinase is not the functional homolog of yeast Ipk2p, i.e. human InsP₄ 5-kinase does not phosphorylate inositol phosphate in a concerted reaction generating InsP₅ from InsP₃. It is formally possible that human InsP₄ 5-kinase could be modulated to exhibit a different substrate profile in the in vivo environment of the human cell. Additionally, we cannot exclude the possibility that other substrates are phosphorylated in vivo. Given the data presented, human InsP₄ 5-kinase likely functions as part of the pathway distal of Ins(1,3,4)P₃ 5/6-kinase and proximal to InsP₅ 2-kinase.

We propose that the synthesis of InsP₆ from Ins(1,4,5)P₃ is more complicated than the sequential phosphorylations seen in the yeast system (Fig. 9). The common precursor Ins(1,4,5)P₃ is synthesized by phospholipase C and then phosphorylated by Ins(1,4,5)P₃ 3-kinase to synthesize Ins(1,3,4,5)P₄ (6). Whether the human InsP₄ 5-kinase can catalyze this 3-kinase step is not clear, especially in the absence of elevated calcium ions where the V_max of Ins(1,4,5)P₃ 3-kinase decreases significantly (6). Next, Ins(1,3,4,5)P₄ is dephosphorylated by 5-phosphatase(s) generating Ins(1,3,4)P₃, which serves as the substrate for Ins(1,3,4)P₃ 5/6-kinase. Phosphorylation at the D-6 position catalyzed by Ins(1,3,4)P₃ 5/6-kinase synthesizes Ins(1,3,4,6)P₄, which is the preferred substrate for Ins(1,3,4,6)P₄ 5-kinase. Phosphorylation at the D-5 position by InsP₄ 5-kinase generates InsP₅; then subsequent phosphorylation at the D-2 position by InsP₅ 2-kinase generates InsP₆. Whether there is another kinase that catalyzes the synthesis of Ins(1,3,4,5)P₄ to InsP₅ remains to be determined.

View larger version (12K): [in this window] [in a new window]   Fig. 9.   Pathway of inositol phosphate synthesis. For details, see "Results." The abbreviations are as follows: InsP3 3-K, Ins(1,4,5)P3 3-kinase; 5-ptase, 5-phosphatase; InsP3 5/6-K, Ins(1,3,4)P3 5/6-kinase; InsP4 5-K, Ins(1,3,4,6)P4 5-kinase; and InsP5 2-K, InsP5 2-kinase.

	ACKNOWLEDGEMENTS

We are grateful to Monita P. Wilson for technical advice in the inositol phosphate field, to John W. Verbsky for the plasmid pLGSD5Trp, and to all members of the Majerus and Wente laboratories for reagents and helpful discussions.

	Addendum

During the preparation of this manuscript, the human homolog of the rat inositol phosphate multikinase was identified by Nalaskowski and co-workers (39).

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants RO1-HL55672 and RO1-HL16634 and Training Grant H107088 (to P. W. M.) and by a Kirsch investigator award from the Steven and Michele Kirsch Foundation (to S. R. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Internal Medicine, 660 S. Euclid Ave., Campus Box 8125, St. Louis, MO 63110. Tel.: 314-362-8801; Fax: 314-362-8826; E-mail: phil@im.wustl.edu.

Published, JBC Papers in Press, September 9, 2002, DOI 10.1074/jbc.M206134200

	ABBREVIATIONS

The abbreviations used are: Ins(1, 3,4,5,6)P₅, inositol 1,3,4,5,6-pentakisphosphate; InsP₆, inositol hexakisphosphate; Ins(1, 4,5)P₃, inositol 1,4,5-trisphosphate; InsP₃ 3-kinase(s), inositol 1,4,5-trisphosphate 3-kinase(s); InsP₃ 5/6-kinase, inositol 1,3,4-trisphosphate 5/6-kinase; Ins(1, 3,4,5)P₄ and Ins(1,3,4,6)P₄, inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4,6-tetrakisphosphate, respectively; InsP₄ 5-kinase, inositol 1,3,4,6-tetrakisphosphate 5-kinase; IPMK, inositol phosphate multikinase; 5-FOA, 5-fluoroorotic acid; HPLC, high performance liquid chromatography; EST, expressed sequence tag; ORF, open reading frame; SAX, strong anion exchange; OCRL, oculocerebrorenal syndrome of Lowe.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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