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J. Biol. Chem., Vol. 275, Issue 32, 24686-24692, August 11, 2000

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The Inositol Hexakisphosphate Kinase Family

CATALYTIC FLEXIBILITY AND FUNCTION IN YEAST VACUOLE BIOGENESIS^*

Adolfo Saiardi^§, James J. Caffrey^§¶, Solomon H. Snyder^**, and Stephen B. Shears^¶

From the Departments of  Neuroscience,  Pharmacology and Molecular Sciences, and ^** Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and the ^¶ Inositide Signaling Section, Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received for publication, March 31, 2000, and in revised form, May 24, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Saiardi et al. (Saiardi, A., Erdjument-Bromage, H., Snowman, A., Tempst, P., and Snyder, S. H. (1999) Curr. Biol. 9, 1323-1326) previously described the cloning of a kinase from yeast and two kinases from mammals (types 1 and 2), which phosphorylate inositol hexakisphosphate (InsP₆) to diphosphoinositol pentakisphosphate, a "high energy" candidate regulator of cellular trafficking. We have now studied the significance of InsP₆ kinase activity in Saccharomyces cerevisiae by disrupting the kinase gene. These ip6k cells grew more slowly, their levels of diphosphoinositol polyphosphates were 60-80% lower than wild-type cells, and the cells contained abnormally small and fragmented vacuoles. Novel activities of the mammalian and yeast InsP₆ kinases were identified; inositol pentakisphosphate (InsP₅) was phosphorylated to diphosphoinositol tetrakisphosphate (PP-InsP₄), which was further metabolized to a novel compound, tentatively identified as bis-diphosphoinositol trisphosphate. The latter is a new substrate for human diphosphoinositol polyphosphate phosphohydrolase. Kinetic parameters for the mammalian type 1 kinase indicate that InsP₅ (K_m = 1.2 µM) and InsP₆ (K_m = 6.7 µM) compete for phosphorylation in vivo. This is the first time a PP-InsP₄ synthase has been identified. The mammalian type 2 kinase and the yeast kinase are more specialized for the phosphorylation of InsP₆. Synthesis of the diphosphorylated inositol phosphates is thus revealed to be more complex and interdependent than previously envisaged.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The very dynamic turnover of the "high energy" diphosphorylated inositol polyphosphates (PP-InsP₄, PP-InsP₅, and [PP]₂-InsP₄)¹ may represent a molecular switching activity that regulates intracellular trafficking (see Ref. 1 for a review). For example, PP-InsP₅ represents the most potent known inhibitor of AP180-mediated assembly of clathrin cages, a key step in the endocytic retrieval of discharged synaptosomal vesicles (2). Other proteins that participate in intracellular trafficking can bind PP-InsP₅ very tightly, including coatomer (3, 4) and AP2 (5). The high affinity with which PP-InsP₅ binds to myelin proteolipid protein may be important for the vesicular delivery of the latter to the myelin sheath (6).

Prior experiments with intact cells have shown that Ins(1,3,4,5,6)P₅ and InsP₆ serve as metabolic stockpiles for the formation of the diphosphorylated inositol polyphosphates (7, 8). InsP₆ is the precursor for PP-InsP₅, which is further phosphorylated to [PP]₂-InsP₄ (5, 7-10). These reactions appear to take place within a metabolic pool that is separate from that in which Ins(1,3,4,5,6)P₅ and PP-InsP₄ are interconverted (7). Thus, there are two metabolic pools of inositol diphosphates that are turned over in parallel cycles.

There are increasing efforts to characterize the activities of the enzymes that regulate the turnover of PP-InsP₄, PP-InsP₅, and [PP]₂-InsP₄. Several phosphatases (diphosphoinositol polyphosphate phosphohydrolases) that hydrolyze these compounds have been described (11, 12). Two forms of InsP₆ kinase (types 1 and 2), derived from distinct genes, have been cloned from mammals (13, 14). At least in mammals, the further phosphorylation of PP-InsP₅ to [PP]₂-InsP₄ appears to be the function of a separate enzyme that has been purified from rat brain (9) but not yet cloned. A yeast InsP₆ kinase has also been cloned that shows approximately 30% sequence similarity to the two mammalian InsP₆ kinases (13). We also recently reported that PP-InsP₅ and [PP]₂-InsP₄ are present in Saccharomyces cerevisiae (15).

A notable omission from our understanding of the intricacies of the turnover of higher inositol phosphates is any characterization of the enzyme(s) that phosphorylate Ins(1,3,4,5,6)P₅ to PP-InsP₄. Thus, an important goal of this study was to identify this "missing link" in inositide research: the PP-InsP₄ synthase. We now report that PP-InsP₄ in mammals is synthesized by the type 1 and type 2 forms of the InsP₆ kinase. In addition, we show that these enzymes can further phosphorylate PP-InsP₄, thereby forming a hitherto unknown inositol polyphosphate.

We have further investigated the activity of the yeast enzyme in several ways. We have characterized its substrate specificity in vitro. In this study, we demonstrate that disrupting the InsP₆ kinase gene of S. cerevisiae (ip6k) influences inositol polyphosphate levels in a unique fashion. We also show that ip6k yeast cells have fragmented vacuoles. These data support an important role for diphosphoinositol polyphosphates in regulating intracellular trafficking.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Enzyme Assays-- The various InsP₆ kinases used in this study were incubated for various times at 37 °C in 25 or 50 µl of buffer containing 20 mM HEPES, pH 7.0, with KOH, 12 mM MgSO₄, 1 mM dithiothreitol, 10 mM ATP, 20 mM phosphocreatine, 1 mM EDTA, 0.02 mg/ml phosphocreatine kinase (Calbiochem 238395) and 0.5 mg/ml bovine serum albumin. The appropriate ³H-labeled inositol phosphate was added as indicated. Assays were quenched with ice-cold perchloric acid and neutralized as described previously (16).

Where specifically indicated, reactions were quenched by incubation for 3 min at 100 °C. Control experiments showed that all kinase activity was inactivated by this heat treatment, but none of the diphosphorylated inositol phosphates were degraded.

HPLC Analyses-- The HPLC analysis of [³H]inositol-labeled yeast cells was performed as described previously (15). Assays of the activities of recombinant enzymes employed HPLC using a Partisphere SAX column (Krackler Scientific, Durham, NC) that was eluted with a gradient generated by mixing Buffer A (1 mM Na₂EDTA) and Buffer B (Buffer A plus 1.3 M (NH₄)₂HPO₄, pH 3.85, with H₃PO₄) as follows: 0-5 min, 0% B; 5-10 min, 0-45% B; 10-60 min, 45-100% B; 60-70 min, 100% B. 1-ml fractions were collected. In some experiments, particularly when a new HPLC column was installed, the percentage of B at 10 min was increased to 50%. This change in the gradient, plus the tendency of inositol phosphates to elute earlier as the column aged, means that slightly different elution properties are seen in the different figures shown in this study.

Preparations of Recombinant Proteins-- Recombinant hDIPP2 was prepared as described previously (11). The cDNAs for mammalian InsP₆ kinase types 1 and 2 were recovered from the pCMV-glutathione S-transferase vector used previously (13). The type 1 kinase cDNA was amplified using the following primers: 5'-GCACTCGAGAATGTGTGTTTGTCAAAC-3' and 5'-GCTAAGCTTAGGGCCTACTGGTTCTC-3'; the polymerase chain reaction product was subcloned into the XhoI and HindIII sites of the pTrcHisB expression vector (Invitrogen). The cDNA for the type 2 kinase was amplified using the following primers: 5'-CGACTCGAGGATGAGCCCAGCCTTCAG-3' and 5'-GCATTCGAAC TCACTCCCCACTCTCCTC-3; the polymerase chain reaction product was subcloned into the XhoI and BstBI restriction sites of pTrcHisB. The methods used to transform Escherichia coli (strain BL21), to induce with isopropyl-1-thio--D-galactopyranoside, and to isolate the poly(His)-tagged proteins using Talon resin (CLONTECH), were all according to the manufacturer's recommendations. The recombinant yeast InsP₆ kinase was produced as described previously (13).

Creation of the ip6k Strain of S. cerevisiae-- The YDR017C open reading frame in S. cerevisiae comprises the kcs1 gene that encodes the yeast InsP₆ kinase. Using strain PJ69-2A of S. cerevisiae, the portion of the kcs1 gene that encodes the C terminus of the kcs1 protein from amino acid 575 to the C-terminal stop codon was replaced by the dominant kan^r marker gene using the KanMX4 expression construct as described previously (17). The oligonucleotides used for the gene disruption were: 5'-GAAGGAAAAGAAACTCTAATACGACTACAATGGGAAACCATAATGCATAGGCCACTAGTGGATCTG-3' and 5'-TAAGCGCAGCTAAAAGAATATTCATTAGTTCTATCCTTTCTTTTCAGCTGAAGCTTCGTACGC-3'.

Northern Analysis of Wild-type and ip6k Yeast-- 10 µg of total RNA prepared from exponentially growing yeast was fractionated on a 1% agarose/MOPS-formaldehyde gel and transferred to Hybond N+ membranes, according to the manufacturer's instructions (Amersham Pharmacia Biotech). The blot was hybridized with a 1.2-kilobase BamHI-NotI fragment corresponding to the 3'-region of the gene. This fragment was obtained by the digestion of the cDNA for the yeast InsP₆ kinase (13).

Vacuole Analyses of Wild-type and ip6k Yeast-- Wild-type and ip6k yeast were grown at 30 °C in YPD medium (20 g/liter peptone, 10 g/liter yeast extract, 2% glucose). Yeasts from 5 ml of early logarithmic phase cultures (A₆₀₀ = 0.4-0.6) were collected and resuspended in 100 µl of medium containing 50 mM sodium citrate buffer, pH 5.0, 2% glucose, 10 µM carboxy-DCFDA. Cells were incubated at room temperature for 20 min. Fluorescence (excitation = 504 nm, emission = 529 nm) was visualized using a Zeiss Axioskop microscope with 100× objective.

Materials-- Nonradioactive InsP₆ and Ins(1,3,4,5,6)P₅ were purchased from Calbiochem (La Jolla, CA) and the Alexis Corporation (San Diego, CA), respectively. Stock solutions were prepared in 1 mM EDTA. Carboxy-DCFDA was purchased from Molecular Probes, Inc. (Eugene, OR). [³H]InsP₆ was purchased from NEN Life Science Products. [³H]Ins(1,3,4,5,6)P₅ and [³H]Ins(3,4,5,6)P₄ were isolated from 5-day-old [³H]inositol-labeled chick erythrocytes (18). [PP]₂-[³H]InsP₄ was obtained by phosphorylation of [³H]InsP₆ using partly purified enzyme preparations from rat brain (19). PP-[³H]InsP₅ and PP-[³H]InsP₄ were prepared by phosphorylation of [³H]InsP₆ and [³H]Ins(1,3,4,5,6)P₅ respectively, using the type 1 InsP₆ kinase (13). ¹⁴C-Labeled Ins(1,3,4,5,6)P₅ was isolated from [¹⁴C]inositol-labeled, parotid acinar glands (20). All of the radiolabeled inositol phosphates that we synthesized were purified by HPLC and desalted (7).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Creation and Analysis of a ip6k Strain of S. cerevisiae-- We recently showed that PP-InsP₅ and [PP]₂-InsP₄ were present in S. cerevisiae (15). One goal of this study was to determine the importance of the yeast InsP₆ kinase to the synthesis of the diphosphorylated inositol phosphates in vivo. The yeast InsP₆ kinase protein is about three times larger in mass than either of the type 1 and type 2 forms of the mammalian InsP₆ kinases (13). The domain of the yeast InsP₆ kinase that is homologous to the smaller, mammalian enzymes resides in a region close to the C terminus. The latter portion of the yeast protein was deleted by disrupting the appropriate 3'-region of the kinase gene (Fig. 1 and see "Experimental Procedures"). To confirm the correct integration of the marker gene, two diagnostic Southern blots were performed. Yeast genomic DNA was digested by SnaBI, and a band of approximately 5.5 kilobases was detected only in the gene-disrupted strain upon hybridization with the kan^r gene (data not shown). Coincidentally, the kan^r cassette used for the gene disruption is almost identical in size to the InsP₆ kinase gene it replaces (Fig. 1). Thus, a band of approximately 5.5 kilobases was detected only in wild-type cells upon hybridization with a probe corresponding to the deleted region of the InsP₆ kinase gene (data not shown). The success of the gene disruption was also verified by Northern analysis of wild-type and mutant yeast (Fig. 1). The gene-disrupted strain is hereafter designated ip6k.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Strategy for the creation of an ip6k strain of S. cerevisiae and Northern blot analysis of WT and ip6k cells. A is a schematic (drawn to scale) that illustrates the domain within the yeast InsP6 kinase into which the KanMX4 cassette was inserted. The light gray box represents the portion of the kinase that is homologous to the mammalian type 1 and type 2 InsP6 kinases. The black box demarcates the area identified by sequence alignments to be a candidate for the active site (13). B is a Northern analysis of the kinase transcript from wild-type (WT) and ip6k yeast. 10 µg of total RNA was loaded in each lane. The blot was hybridized with a probe corresponding to the 3' portion of the yeast kinase gene that was replaced by the KanMX4 cassette in the ip6k strain (see under "Experimental Procedures"). The arrows indicate the migration of the 25 S and 18 S ribosomal RNA. C shows the results of ethidium bromide staining of the gel used in B to check equivalence of loading.

We investigated the consequences of the gene disruption upon the inositol phosphate profile of wild-type and ip6k cells. Levels of PP-InsP₅ in ip6k cells were >80% lower than those of wild-type cells (Table I). This result demonstrates that this kinase is quantitatively important for the expression of PP-InsP₅ synthase activity in vivo. Nevertheless, our results indicate that there must be an alternative, hitherto unrecognized, pathway for PP-InsP₅ synthesis in yeast. Levels of [PP]₂-InsP₄ were 60% lower in the ip6k cells compared with the wild type (Table I). This result indicates that the InsP₆ kinase plays only a partial role in the pathway of [PP]₂-InsP₄ synthesis. Note also that the levels of Ins(1,3,4,5,6)P₅ in the ip6k cells were 80% lower than those of wild-type cells (Table I). Thus, in yeast, there is an unexpected link between the metabolism of PP-InsP₅ and Ins(1,3,4,5,6)P₅.

View this table: [in this window] [in a new window]   Table I Comparison of levels of higher inositol phosphates in wild-type and ip6k strains of S. cerevisiae Levels of inositol phosphates were determined by HPLC as described under "Experimental Procedures." Data are the means and S.E. from three experiments. Levels of the less phosphorylated inositol phosphates (InsPn, where n = 1-4) were similar in both wild-type (WT) and ip6k cells.

The ip6k cells grew more slowly than the wild-type cells at 23 and 30 °C (Fig. 2). At 37 °C, the ip6k cells did not grow at all (Fig. 2). Prior to the identification of the yeast InsP₆ kinase (13), the gene that encodes this protein was known as kcs1 (21). In that earlier study, the deletion of the kcs1/InsP₆ kinase gene from S. cerevisiae yielded no apparent growth phenotype (21), possibly because that strain of yeast had a different genetic background from the strain that we have used.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Growth phenotype of wild-type and ip6k strains of S. cerevisiae. The data represent growth curves in liquid cultures of wild-type (closed symbols, solid lines) and ip6k (open symbols, broken lines) yeast strains. A single colony was inoculated in 50 ml of YPD medium and grown at one of the following temperatures: 23 °C (circles), 30 °C (squares), or 37 °C (triangles). Growth was monitored by measuring the optical density of the cultures at 600 nm at the indicated times after inoculation.

Nuclear mRNA Export Is Not Impaired in ip6k Cells-- We (15) and others (22) have previously shown that drastic impairment of the pathway of InsP₆ synthesis in S. cerevisiae is accompanied by a decreased efficiency in the rate of export of mRNA from the nucleus. We (15) did point out, however, that mRNA export may be regulated by metabolites of InsP₆ (such as PP-InsP₅ and [PP]₂-InsP₄) rather than by InsP₆ itself. Indeed, we previously showed that one particular gene-disrupted strain of yeast (ipmk), which displayed an impaired mRNA export phenotype and decreased synthesis of InsP₆, also had 60-80% lower levels of PP-InsP₅ and [PP]₂-InsP₄ (15). The ip6k cells gave us the first opportunity to study whether mRNA export was affected by quantitatively similar changes in levels of PP-InsP₅ and [PP]₂-InsP₄, under conditions where InsP₆ levels were not significantly affected (Table I). We measured mRNA export as described previously (15) but found no significant difference between wild-type and ip6k cells (data not shown). This negative result redirected our efforts to the hypothesis (see the Introduction) that diphosphoinositol polyphosphates regulate protein trafficking.

Altered Vacuolar Morphology in ip6k Cells-- In yeast cells, several different vesicle transport pathways converge upon the vacuole (23). This organelle receives endocytic traffic from the cell surface as well as biosynthetic traffic from the Golgi apparatus (23, 24). Thus, we studied the effect of the deletion of the InsP₆ kinase upon vacuole morphology. The luminal interior of yeast vacuoles was identified by incubating cells with membrane-permeable carboxy-DCFDA (25). Once the probe has entered the vacuoles, the acetate groups are hydrolyzed by nonspecific esterases, forming the less membrane permeable and fluorescent carboxy-DCF (25). Fluorescence detection of the vacuolar space in wild-type cells revealed the usual complement (26) of one large vacuole (Fig. 3). In contrast, the ip6k strain contained several smaller, fragmented vacuoles (Fig. 3). This altered vacuolar morphology may reflect defects in the fusion into vacuoles of small vesicles derived from endocytosis or from the trans-Golgi network. The intensity of staining is also decreased in the null strain (Fig. 3), possibly because of less active intraluminal esterase activity in the ip6k cells.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Fluorescence detection of the vacuolar lumen in wild-type and ip6k strains of S. cerevisiae. Early logarithmic phase cultures of wild-type (WT) and ip6k yeast were incubated with carboxy-DCFDA as described under "Experimental Procedures." Fluorescence detection of the carboxy-DCF that accumulates in the vacuolar space was performed as described under "Experimental Procedures."

Substrate Specificity of the Yeast InsP₆ Kinase-- Could the lower levels of [PP]₂-InsP₄ in the ip6k yeast cells (Table I) reflect an unexpected ability of the yeast InsP₆ kinase to phosphorylate PP-InsP₅? To examine this question, we compared the rates of phosphorylation of both InsP₆ and PP-InsP₅ by the recombinant yeast InsP₆ kinase. Trace amounts of [³H]InsP₆ were almost completely phosphorylated to PP-[³H]InsP₅ (Fig. 4A and Ref. 13). The yeast kinase was also separately incubated with PP-[³H]InsP₅, but only approximately 5% of the substrate was converted to [PP]₂-InsP₄ (Fig. 4C), and in any case, even this slow reaction required 5-fold higher concentrations of enzyme than were used for the assays of InsP₆ phosphorylation (Fig. 4A). Furthermore, levels of InsP₆ in S. cerevisiae are 180-fold higher than those of PP-InsP₅ (Table I). These data indicate that the yeast InsP₆ kinase does not provide an efficient route for [PP]₂-InsP₄ synthesis in vivo. We must therefore search for a separate yeast PP-InsP₅ kinase with greater catalytic efficiency that can account for [PP]₂-InsP₄ synthesis in vivo.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Substrate specificity of yeast InsP6kinase. The recombinant yeast InsP6 kinase was incubated as described under "Experimental Procedures" for either 0 min (open circles) or for the times indicated below (filled circles). In A, 3 pg/µl kinase was incubated for 1 h. with approximately 12,000 dpm of [3H]InsP6. In B, 13 pg/µl kinase was incubated for 1 h with approximately 15,000 dpm of [3H]Ins(1,3,4,5,6)P5. In C, 14 pg/µl kinase was incubated for 2 h with approximately 600 dpm of PP-[3H]InsP5. Assays were analyzed by HPLC as described under "Experimental Procedures." These data are representative of three experiments.

We also found that the yeast InsP₆ kinase phosphorylated Ins(1,3,4,5,6)P₅ (Fig. 4B). The V_max values for InsP₅ and InsP₆ were each approximately 2 µmol/mg/min. The affinity of the enzyme for InsP₆ (mean K_m = 3.3 µM) was about 3-fold less than the affinity for InsP₅ (mean K_m = 1.2 µM). This is the first time an enzyme with PP-InsP₄ synthase activity has been identified. In the case of S. cerevisiae, cellular levels of InsP₆ are 50-fold higher than those of InsP₅ (Table I), so we would not anticipate substantial phosphorylation of InsP₅ by this kinase in intact yeast cells. However, in mammalian cells, levels of Ins(1,3,4,5,6)P₅ and InsP₆ are very similar to each other (27). We therefore next investigated whether either of the two mammalian InsP₆ kinases (named types 1 and 2; see Ref. 13) could also phosphorylate Ins(1,3,4,5,6)P₅.

Phosphorylation of InsP₅ by the Mammalian Type 1 InsP₆ Kinase-- The type 1 InsP₆ kinase was expressed in E. coli as a His-tagged protein and purified using Talon resin (Fig. 5). The enzyme was incubated with trace amounts of [³H]InsP₆ and an ATP regeneration system (see "Experimental Procedures"). The InsP₆ was found to be completely phosphorylated to PP-[³H]InsP₅ (Fig. 6A and Ref. 13). No [PP]₂-InsP₄ was formed (data not shown and see Ref. 13). Several other inositol polyphosphates, namely inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate, have also previously been found not to be significant substrates for this enzyme (13). However, Ins(1,3,4,5,6)P₅ was not previously tested as a substrate. We now studied this issue, in view of the observation that the yeast kinase phosphorylated both InsP₆ and Ins(1,3,4,5,6)P₅ (Fig. 4).

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Purity of recombinant mammalian InsP6 kinases. Either 25 ng of the purified, recombinant type 1 InsP6 kinase (lane 1) or 10 ng of the purified, recombinant type 2 InsP6 kinase (lane 2) were loaded on a 4-12% polyacrylamide Bis-Tris NuPage gel (Novex, San Diego CA) and electrophoresed with MES-SDS running buffer. The gel was stained with Coomassie Blue. The molecular mass standards are indicated.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Phosphorylation of [3H]InsP6 and [3H]InsP5 by the mammalian type 1 InsP6 kinase. All incubations were performed as described under "Experimental Procedures." A depicts an HPLC analysis of reactions containing the type 1 kinase (1 ng/µl) incubated with approximately 5000 dpm of [3H]InsP6 for either 0 min (open circles) or 120 min (closed circles). B depicts an HPLC analysis of reactions containing the type 1 kinase (2 ng/µl) incubated for 0 min (open circles) or 120 min (closed circles) with approximately 10,000 dpm of [3H]Ins(1,3,4,5,6)P5. Peak X is a previously unknown product that we have identified as [PP]2-InsP3 (see text). The inset to B shows a time course for the phosphorylation of Ins(1,3,4,5,6)P5 (open circles) to both PP-[3H]InsP4 (closed circles) and peak X (squares) by 2 ng/µl of the kinase. These data are representative of three experiments.

The type 1 InsP₆ kinase phosphorylated Ins(1,3,4,5,6)P₅ (Fig. 6B). The V_max values for Ins(1,3,4,5,6)P₅ and InsP₆ were very similar (Table II). The affinity of this enzyme for InsP₆ was only 5-fold higher than that for Ins(1,3,4,5,6)P₅ (Table II). Because levels of both InsP₆ and Ins(1,3,4,5,6)P₅ in mammalian cells each range from 15 to 50 µM (27), we can anticipate that these two substrates will compete for phosphorylation by this enzyme in vivo.

InsP₅ Is Converted to a Novel Inositol Polyphosphate by Mammalian Type 1 InsP₆ Kinase-- Another unexpected result to emerge from the studies of InsP₅ phosphorylation by the type 1 mammalian kinase was the accumulation of a novel product (Fig. 6B, peak X), at a rate that was approximately 10% of the rate of accumulation of PP-InsP₄ (Fig. 6B, inset). A comparison of panels A and B in Fig. 6 also shows that the elution position of peak X was between those of InsP₆ and PP-InsP₅.

We considered the possibility that X represents one of two different PP-InsP₄ isomers, each formed by direct phosphorylation of InsP₅. This option seemed unlikely, because in our HPLC experiments, peak X eluted 10 min later than did PP-InsP₄ (Fig. 6B); this HPLC system can only resolve isomers of diphosphoinositol polyphosphates by 1-2 min even when a considerably more shallow elution gradient is employed (5). Furthermore, when the kinase was separately incubated with PP-[³H]InsP₄, approximately 50% of this substrate was phosphorylated to peak X (Fig. 7). In other words PP-InsP₄, and not InsP₅, is the immediate precursor of peak X. 

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Phosphorylation of PP-[3H]InsP4 by the mammalian type 1 InsP6 kinase. The figure shows an HPLC analysis of assays (performed as described under "Experimental Procedures") containing the type 1 kinase (2 ng/µl) incubated with approximately 5500 dpm of PP-[3H]InsP4 for either 0 min (open circles) or 120 min (closed circles). These data are representative of three experiments.

We next considered whether peak X might be another isomer of PP-InsP₅. This explanation also seems an unlikely possibility, because the synthesis of PP-InsP₅ from PP-InsP₄ would not involve the formation of a diphosphate group. Nevertheless, we checked the nature of peak X by using, as a diagnostic tool, one of the several hDIPP isoforms that we have cloned (hDIPP2) (11). The hDIPP2 enzyme specifically removes -phosphates from diphosphorylated inositol polyphosphates; hDIPP2 does not hydrolyze monoester phosphates (11). When hDIPP2 was incubated with a mixture of PP-InsP₄ and peak X, both were completely dephosphorylated back to InsP₅ (Fig. 8). Thus, peak X cannot be PP-InsP₅, because the latter is dephosphorylated to InsP₆ by hDIPP2 (11).

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Dephosphorylation of PP-[3H]InsP4 and peak X by hDIPP2. A mixture of PP-[3H]InsP4 and 3H-labeled peak X was prepared as described in the legend to Fig. 7, except that assays were quenched by exposure of the reaction tubes to 100 °C for 3 min. The tubes were cooled on ice and then divided into two equal aliquots which received either vehicle (open circles) or 1.9 µg of purified hDIPP2 (closed circles). Reaction tubes were incubated for a further 2 h at 37 °C, and these assays were finally acid-quenched and analyzed by HPLC as described under "Experimental Procedures." The upper panel shows the profile of 3H-labeled material. The lower panel shows the elution position of a [14C]InsP5 standard, which was determined in a parallel HPLC run. These data are representative of three experiments.

We therefore propose that peak X may be a hitherto unknown, doubly diphosphorylated derivative of InsP₅, namely, [PP]₂-InsP₃. The [PP]₂-InsP₃ would be analogous to [PP]₂-InsP₄, which is the doubly diphosphorylated derivative of InsP₆ (Fig. 9).

View larger version (12K): [in this window] [in a new window]   Fig. 9.   Synthesis of diphosphorylated inositol polyphosphates. The figure shows the metabolic pathway for the synthesis of diphosphorylated inositol polyphosphates. The solid arrows indicate those catalytic steps that are performed by the inositol hexakisphosphate kinases. PP-InsP5 is the only diphosphorylated inositol phosphate for which the position of the diphosphate group on the inositol ring has been clarified (it is attached to the 5-carbon (37)).

Although PP-InsP₄ is a substrate for the type-1 kinase, this enzyme was very ineffective at further phosphorylating the PP-InsP₅ that was formed from InsP₆ (Fig. 4, A and C). The difference between PP-InsP₅ and PP-InsP₄ lies in the latter having a 2-OH instead of a 2-phosphate group, and this clearly has a substantial impact upon substrate specificity.

Comparisons between the Type 1 and Type 2 Mammalian InsP₆ Kinases-- The amino acid sequences of the mammalian type 1 and type 2 InsP₆ kinases are about 60% similar (13). We therefore compared the activities of these two enzymes in more detail. We found that the purified, recombinant type 2 kinase (Fig. 5) was able to phosphorylate both InsP₅ and InsP₆ (Table II). In kinetic experiments, we established that V_max values for both Ins(1,3,4,5,6)P₅ and InsP₆ were very similar (Table II). The affinity of the type 2 kinase for Ins(1,3,4,5,6)P₅ (K_m = 8.4 µM, Table II) was approximately 20-fold lower than the affinity for InsP₆ (K_m = 0.43 µM, Table II). These data therefore identify an important difference between the two kinases, namely, that Ins(1,3,4,5,6)P₅ will compete with InsP₆ much less effectively for phosphorylation by the type 2 kinase, compared with the type 1 enzyme.

In view of the ability of the mammalian InsP₆ kinases to phosphorylate Ins(1,3,4,5,6)P₅, we examined whether Ins(3,4,5,6)P₄ was a substrate. Ins(3,4,5,6)P₄ is a cellular signal that regulates the conductance of Ca²⁺-activated chloride channels (28, 29), so the understanding of the metabolism of this inositol phosphate is a topic of some importance. We found that for both the type 1 and type 2 kinases, Ins(3,4,5,6)P₄ was a 40-50-fold weaker substrate compared with Ins(1,3,4,5,6)P₅ (Table III). This observation and our knowledge that cellular levels of Ins(1,3,4,5,6)P₅ are 5-10-fold higher than those of Ins(3,4,5,6)P₄ (27) lead us to conclude that there will not be significant phosphorylation of Ins(3,4,5,6)P₄ by this route in vivo. Nevertheless, these data usefully indicate that the 1-phosphate is important in determining substrate specificity of the InsP₆ kinases (Table III).

We also compared the rates at which the two kinases phosphorylated PP-InsP₄ to [PP]₂-InsP₃. We could not prepare sufficient mass amounts of PP-InsP₄ to ascertain K_m and V_max values. Instead we compared rates of PP-InsP₄ phosphorylation under first order conditions. The activity of the type 2 kinase toward PP-InsP₄ was approximately 7-fold lower than the activity of the type 1 kinase (Table III). The two enzymes showed only a 2-fold difference in activity toward Ins(1,3,4,5,6)P₅ (Table III).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study we have obtained important new information concerning the physiological significance of the InsP₆ kinase family. We have discovered that Ins(1,3,4,5,6)P₅ can also be phosphorylated by these enzymes (Fig. 9), and we have obtained data indicating that the yeast InsP₆ kinase plays an important role in vacuole biogenesis (Fig. 3).

Ins(1,3,4,5,6)P₅ and PP-InsP₄ have previously been allocated to a metabolic pool that is different from the pool in which InsP₆, PP-InsP₅, and [PP]₂-InsP₄ are contained (7). It would, therefore, not have been surprising if the turnover of these two metabolically distinct groups of compounds had turned out to be independently regulated and also functionally discrete. As a result of the work described in the current study, we can now appreciate for the first time just how interdependent are these metabolic cycles, in mammalian cells at least (Fig. 9). Levels of InsP₆ and Ins(1,3,4,5,6)P₅ in mammalian cells range from 15 to 50 µM (27), and in some cells Ins(1,3,4,5,6)P₅ levels are 4-fold higher than those of InsP₆ (7). This information, together with the new data on the kinetic parameters for Ins(1,3,4,5,6)P₅ and InsP₆ phosphorylation by mammalian type 1 InsP₆ kinase (Table II), indicates that both of these substrates will compete for phosphorylation by this enzyme in vivo. If the size of the metabolic reservoir of either substrate is modified, as happens for example during passage through the cell cycle (30, 31), the metabolism of all of the diphosphorylated compounds will be affected. From this intertwined metabolism, we can further conclude that the physiological activities of all of the diphosphorylated inositol phosphates are likely to be closely linked.

Our studies also provide new insight into the structural determinants of InsP₆ kinase specificity. With regards to the mammalian type 1 InsP₆ kinase, the V_max and K_m values were similar for phosphorylation of both Ins(1,3,4,5,6)P₅ and InsP₆ (Table II). Thus, for these two substrates, there is only a minor impact on the catalytic activity of this enzyme when a phosphate group substitutes for a hydroxyl group at the 2-position on the inositol ring. On the other hand, PP-InsP₄ can be further phosphorylated much more readily than can PP-InsP₅ (Figs. 6 and 7). The difference between PP-InsP₄ and PP-InsP₅ is that the latter has a 2-phosphate, which in this context imposes an important constraint upon catalytic activity. Thus, the impact of the 2-phosphate group on enzyme activity is critically dependent upon the nature of the substrate. Note also that two very weak substrates for the kinases are Ins(3,4,5,6)P₄ (Table III) and inositol 1,3,4,5-tetrakisphosphate (13), so phosphate groups at both the 1- and 6-positions are also very important to substrate recognition.

Our identification of a novel diphosphorylated inositol phosphate (Fig. 6, peak X) may also provide an unexpected new direction for future research into this area. This compound, tentatively identified as [PP]₂-InsP₃, was synthesized by both mammalian InsP₆ kinases (Table III), and by the yeast InsP₆ kinase (data not shown), suggesting that the catalytic site of these enzymes has not had some flexibility in the positions on the inositol ring at which the diphosphate groups are added. The mammalian type 1 kinase had the most active [PP]₂-InsP₃ synthase activity (Table III). It would therefore be useful to ascertain whether [PP]₂-InsP₃ can be detected in vivo. However, the identification of any of the diphosphorylated inositol phosphates inside intact cells is problematic, because their steady-state levels are so low. Indeed, these compounds escaped detection for several years, even after the discovery of Ins(1,3,4,5,6)P₅ and InsP₆ inside animal cells (32, 33). To date, we have not detected any significant levels of [PP]₂-InsP₃ in HPLC analyses of extracts of [³H]inositol-labeled rat AR42J cells (7), primary cultured rat hepatocytes (34), or hamster DDT₁ MF-2 smooth muscle cells (35). However, this search has so far only involved a small number of cell types. Additionally, the metabolism of the diphosphorylated inositol phosphates is carefully regulated (1), and so we may not have incubated cells under the appropriate conditions. By analogy, it is worth noting that it was some years after the identification of PtdIns(4,5)P₂ before the more minor lipids such as PtdIns(3,4,5)P₃ and PtdIns(3,5)P₂ were detected and found to be significant (36).

Our studies have also provided new insights into the activities of the yeast InsP₆ kinase in vivo. The persistence of significant levels of diphosphorylated inositol phosphates in ip6k cells (Table I) indicates that the InsP₆ kinase we have studied plays a major, but not an exclusive, role in the synthesis of this group of compounds in vivo. Thus, we need to search for an independent (albeit more minor) metabolic pathway that yeast can use to synthesize PP-InsP₅. Moreover, this alternative pathway can sustain a certain amount of [PP]₂-InsP₄ synthesis (Table I). The newly discovered competition of Ins(1,3,4,5,6)P₅ and InsP₆ for phosphorylation by the InsP₆ kinase, also speaks to a previously puzzling observation that cellular levels of PP-InsP₄ increased dramatically in yeast when the Ins(1,3,4,5,6)P₅ 2-kinase activity was deleted (22). Because the elimination of the Ins(1,3,4,5,6)P₅ 2-kinase was accompanied by loss of cellular InsP₆ (22), we can now appreciate that this would have removed an inhibitory constraint upon the conversion of Ins(1,3,4,5,6)P₅ to PP-InsP₄. Another interesting phenomeonon in the ip6k cells was their dramatically (approximately 80%) reduced levels of Ins(1,3,4,5,6)P₅ compared with wild-type cells (Table I). Understanding the molecular mechanisms that link Ins(1,3,4,5,6)P₅ metabolism to InsP₆ kinase activity will be an interesting topic for future studies. At the very least, we should be cognizant that the deletion of the InsP₆ kinase has unexpected repercussions that may contribute to the growth-impaired phenotype of ip6k cells.

In addition to the decrease in PP-InsP₅ levels (Table I), the ip6k yeast contain abnormally small and fragmented vesicles (Fig. 3). Yeast vacuoles are homologues of mammalian lysosomes, possessing acidic interiors where protein degradation takes place (23). The vacuoles derive from fusion of cytoplasm-derived vesicles as well as clathrin-coated vesicles associated with the endocytic apparatus or the trans-Golgi network (23, 24). PP-InsP₅ binds tightly to adaptor proteins that help assemble both clathrin-coated and non-clathrin-coated vesicles (2-5). Thus, the altered vacuolar morphology in the ip6k yeast may reflect some abnormality in the inositol polyphosphate-dependent assembly of those vesicles that normally form vacuoles.

By determining the structural and functional relationships that exist between proteins from different organisms, we can gain insight into their evolutionary origins and physiological roles. Clearly, the catalytic activities of the yeast and mammalian InsP₆ kinases are all rather similar, suggesting that there has been evolutionary conservation of their active site domains. The ability of Ins(1,3,4,5,6)P₅ to compete with InsP₆ for phosphorylation by these kinases does differ between the type 1 and type 2 mammalian kinases. This is suggestive of some divergence in the function of these two proteins. This conclusion is pertinent to understanding the physiological consequences that might arise from tissue-dependent differences in the expression of the type 1 and 2 isoforms, as has been described in the rat, for example (13). Overall, the dual specificity of the type-1 kinase toward Ins(1,3,4,5,6)P₅ and InsP₆ has uncovered an unexpectedly close metabolic and functional linkage between the turnover of all of the diphosphorylated inositol phosphates.

	ACKNOWLEDGEMENTS

We thank Bevery Wendland and Eiichiro Nagata for helpful discussions and Adele Snowman for great technical assistance. Supported by USPHS grant MH18501 (SHS) and Research Scientist Award DA00074 (SHS).

	FOOTNOTES

^* 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.

^§ These authors contributed equally to this work.

To whom correspondence should be addressed: Inositide Signaling Group, NIEHS, 111 Alexander Dr., Research Triangle Park, NC 27709. Tel.: 919-541-0793; Fax: 919-541-0559; E-mail: Shears@niehs.nih.gov.

Published, JBC Papers in Press, May 27, 2000, DOI 10.1074/jbc.M002750200

	ABBREVIATIONS

The abbreviations used are: PP-InsP₄, diphosphoinositol tetrakisphosphate; PP-InsP₅, diphosphoinositol pentakisphosphate; [PP]₂-InsP₃, bis-diphosphoinositol trisphosphate; [PP]₂-InsP₄, bis-diphosphoinositol tetrakisphosphate; carboxy-DCFDA, 5- (and 6-)carboxy-2',7'-dichlorofluorescein diacetate; carboxy-DCF, 5- (and 6-)carboxy-2',7'-dichlorofluorescein; hDIPP, human diphosphoinositol polyphosphate phosphohydrolase; Ins(3, 4,5,6)P₄, D-myo-inositol 3,4,5,6-tetrakisphosphate; InsP₆, inositol hexakisphosphate; InsP₅, inositol pentakisphosphate; Ins(1, 3,4,5,6)P₅, D-myo-inositol 1,3,4,5,6-pentakisphosphate; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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