©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Synthesis and Metabolism of Bis-diphosphoinositol Tetrakisphosphate in Vitro and in Vivo(*)

Stephen B. Shears (1)(§), Nawab Ali (1), Andrew Craxton (1), Micheal E. Bembenek (2)

From the (1) Inositol Lipid Section, Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 and the (2) NEN Medical Products Department, E. I. Du Pont Nemours & Co (Inc.), Boston, Massachusetts 02118

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The pathway of synthesis and metabolism of bis-diphosphoinositol tetrakisphosphate (PP-InsP-PP) was elucidated by high performance liquid chromatography using newly available H- and P-labeled substrates. Metabolites were also identified by using two purified phosphatases in a structurally diagnostic manner: tobacco ``pyrophosphatase'' (Shinshi, H., Miwa, M., Kato, K., Noguchi, M. Matsushima, T., and Sugimura, T. (1976) Biochemistry 15, 2185-2190) and rat hepatic multiple inositol polyphosphate phosphatase (MIPP; Craxton, A., Ali, N., and Shears, S. B. (1995) Biochem. J. 305, 491-498). The demonstration that diphosphoinositol polyphosphates were hydrolyzed by MIPP provides new information on its substrate specificity, although MIPP did not metabolize significant amounts of these polyphosphates in either rat liver homogenates or intact AR4-2J cells. In liver homogenates, inositol hexakisphosphate (InsP) was phosphorylated first to a diphosphoinositol pentakisphosphate (PP-InsP) and then to PP-InsP-PP. These kinase reactions were reversed by phosphatases, establishing two coupled substrate cycles. The two dephosphorylations were probably performed by distinct phosphatases that were distinguished by their separate positional specificities, and their different sensitivities to inhibition by F (IC values of 0.03 mM and 1.4 mM against PP-InsP and PP-InsP-PP, respectively). In [H]inositol-labeled AR4-2J cells, the steady-state levels of PP-[H]InsP and PP-[H]InsP-PP were, respectively, 2-3 and 0.6% of the level of [H]InsP. The ongoing turnover of these polyphosphates was revealed by treatment of cells with 0.8 mM NaF for 40 min, which reduced levels of [H]InsP by 50%, increased the levels of PP-[H]InsP 16-fold, and increased levels of PP-[H]InsP-PP 5-fold. A large increase in levels of PP-[H]InsP also occurred in cells treated with 10 mM NaF, but then no significant change to levels of PP-[H]InsP-PP were observed; there may be important differences in the control of the turnover of these two compounds.


INTRODUCTION

The extensive literature on the synthesis, metabolism, and functions of inositol phosphates is largely focused on Ins (1, 4, 5) P,() Ins (1, 3, 4, 5) P, and the pathways by which these compounds are recycled to inositol (1, 2) . There is a substantially smaller body of information on the more highly phosphorylated inositol phosphates such as InsP. Interest in this particular inositol polyphosphate has now increased following the discovery that it is further phosphorylated to a diphosphoinositol pentakisphosphate (PP-InsP) (3, 4) , a widely distributed cellular constituent of organisms as phylogenetically diverse as Dictyostelium discoideum(4) and rat (3) . PP-InsP has added a new dimension to the study of the many aspects of cell biology that are regulated by a variety of inositol derivatives.

The search for the physiological significance of PP-InsP is currently taking several divergent lines of enquiry. One area of interest in our laboratory is to understand the consequences of high affinity binding of PP-InsP to AP-2,() AP-3 (5) and coatomer (6) . These three proteins are functionally related by virtue of their participation in the control of vesicle traffic (7, 8) ; this important cellular activity is therefore a candidate for a site of action of PP-InsP. Indeed, PP-InsP is the most potent, known inhibitor of AP-3-dependent clathrin assembly (5) . This raises the possibility that polyphosphates such as PP-InsP could represent one of the series of ``fusion clamps'' which may need to be released before the synaptic vesicle can proceed rapidly through its exocytic-endocytic cycle (7, 8) .

Other studies have also provided important information about the metabolic characteristics of PP-InsP; it is particularly striking that in intact mammalian cells there is an ongoing, rapid metabolic flux through the kinase-phosphatase cycle that interconverts InsP with PP-InsP(3) . The high turnover of PP-InsP was one of the factors which led to the suggestion that it might be an important energy donor (4) . Another interesting finding is that, in intact cells, levels of PP-InsP decreased following treatment with thapsigargin (9) . It appears that the InsP kinase was inhibited by thapsigargin-mediated changes in the status of cellular Ca pools (9, 10) . The regulation of PP-InsP turnover by second messengers such as Ca is further testimony to the likely importance of this compound.

In contrast to the catalogue of exciting observations concerning PP-InsP, there is another diphosphate derivative of InsP in mammalian cells (3, 4) that has not yet been studied in any detail. It is possible that this is a bis-diphosphoinositol tetrakisphosphate (PP-InsP-PP); such material has been isolated from Dictyostelium(4) , and it has HPLC elution properties very similar to the mammalian compound. We are unlikely to appreciate the physiological significance of these diphosphoinositol polyphosphates until ( a) we determine how many members of this class of compounds are present in cells, and ( b) we understand all their metabolic characteristics and inter-relationships. With these objectives in mind, we have conducted the first detailed study of the synthesis and metabolism of PP-InsP-PP in vitro and in vivo.

The discovery of diphosphoinositol polyphosphates has also provided us with the opportunity to gain further insight into a puzzling enzyme activity which was originally identified as an Ins (1, 3, 4, 5) P 3-phosphatase (11) . The demonstration that this enzyme also hydrolyzed Ins (1, 3, 4, 5, 6) P and InsP has led to its redesignation as a multiple inositol polyphosphate phosphatase (MIPP) (12, 13) . We have now sought further information on the specificity of this intriguing enzyme by investigating whether diphosphoinositol polyphosphates may also be substrates. Additionally, these experiments have provided further confirmation of the structures of the diphosphoinositol polyphosphates.


EXPERIMENTAL PROCEDURES

Materials

MIPP, originally designated as ``Ins (1, 3, 4, 5) P 3-phosphatase,'' was purified from rat liver (13) . [H]Inositol was obtained from American Radiolabeled Chemicals (St. Louis, MO). A H-labeled diphosphoinositol tetrakisphosphate was prepared as described previously (3) . The following were obtained from DuPont NEN (catalogue numbers are in parentheses): NENSORBpreparative deproteinizing cartridges (NLP 028), [H]InsP (NET 1023), PP-[H]InsP (NET 1093), PP-[H]InsP-PP (NET 1098), [-P]PP-InsP (NEG 214), and [-P]PP-InsP-PP (NEG 215). For some experiments, PP-[H]InsP and PP-[H]InsP-PP were prepared by phosphorylation of [H]InsP and PP-[H]InsP respectively, using liver homogenate incubated as described under ``Experimental Procedures.'' There were no differences in the results obtained using substrates provided by NEN, as compared to those synthesized at NIEHS. Non-radioactive InsP was from Aldrich. Bovine serum albumin, Triton X-100, NaEDTA, and tobacco pyrophosphatase were obtained from Sigma. NaF was obtained from either Sigma or Fluka.

Preparation of Liver Homogenate

Sprague-Dawley rats (200-250 g) were asphyxiated with CO, and blood was removed from the liver by perfusion with ice-cold buffer containing 300 mM sucrose, 1 mM EGTA, 1 mM dithiothreitol, and 10 mM HEPES (pH 7.2 with KOH). The liver was placed in 25 ml of perfusion buffer supplemented with 15 µg/ml leupeptin, 0.2 µg/ml aprotinin, and 15 µM trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane. The liver was chopped with scissors, gently homogenized with a motor-driven Teflon pestle, and filtered through cheesecloth; protein concentration was measured (14) with bovine serum albumin as a standard. Homogenates were fractionated into soluble and particulate fractions by centrifugation for 1 h at 100,000 g; each fraction was adjusted to the same volume as the parent homogenate with homogenization buffer.

Inositol Polyphosphate Metabolism

For the study of the phosphorylation of inositol polyphosphates, trace amounts of radiolabeled substrate were incubated at 37 °C in 0.5-1-ml aliquots of assay buffer 1 (100 mM KCl, 7 mM MgSO, 5 mM ATP, 10 mM phosphocreatine, 10 mM NaF, 10 mM HEPES (pH 7.2 with KOH), 1 mM NaEDTA, 1 mM dithiothreitol, and 0.05 mg/ml phosphocreatine kinase). Dephosphorylation of inositol polyphosphates was studied using assay buffer 2 (50 mM KCl, 50 mM HEPES (pH 7.2 with KOH), 4 mM CHAPS, 0.05 mg/ml bovine serum albumin, 1 mM NaEDTA, 2 mM MgSO). The reactions were initiated by the addition of aliquots of appropriately diluted tissue and quenched with 4 ml of ice-cold medium comprising 0.1 M Tris (pH 7.7 with HCl at 25 °C), 10 mM triethylamine, 2 mM NaEDTA. Protein was subsequently removed in the following manner: a NENSORBpreparative deproteinizing cartridge (15) was first primed with 10 ml of methanol, followed by 10 ml of quench buffer. The quenched reaction was added to the column, which was then washed with 2 4-ml aliquots of quench medium. The flow-rate was 3 ml/min. Recoveries of inositol polyphosphates exceeded 95% in the ``flow-through'' from the column, which was analyzed by HPLC (see below) either immediately, or following storage at -20 °C. Alternately, when metabolism was to be analyzed by gravity-fed anion-exchange columns (see below), assays were quenched with 0.2 ml of 2 M HClO and then neutralized with KOH (16) .

The metabolism of inositol polyphosphates by tobacco ``pyrophosphatase'' (17) was performed by a modification of earlier procedures (3) . Trace amounts of H-labeled substrate were incubated at 37 °C with 100-300 µl of medium containing 30 mM sodium acetate buffer (pH 5.5), 10 mM NaEDTA, 2 mM dithiothreitol, 0.01% (v/v) Triton X-100, 0.4 mg/ml bovine serum albumin. Immediately prior to use, the stock solution of pyrophosphatase was diluted 1:1 with 50 mM NaEDTA (pH 7.0) and added to the incubation buffer to a final concentration of 150-170 Sigma Units/ml. After 16-22 h, incubations were quenched with 4 volumes of ice-cold water, followed by 2 volumes of 0.2 ml of 2 M perchloric acid plus 1 mg/ml InsP. Samples were neutralized with freon-octylamine (16) . There was no hydrolysis of substrate in control samples to which enzyme was not added.

Analysis of Inositol Polyphosphate Metabolism by Gravity-fed Anion-exchange Columns

The release of [P]P from P-labeled diphosphoinositol polyphosphates was conveniently assayed by loading acid-quenched, KOH-neutralized samples (see above) in 10 ml of water, onto 0.8 ml of gravity-fed anion-exchange columns (Bio-Rad, AG1-X8 200-400 mesh, formate form). [P]P was eluted with 2 10-ml aliquots of 0.4 M ammonium formate, 0.1 M formic acid. The diphosphoinositol polyphosphate (recovery = 83 ± 1%, n = 11) was then eluted with 2 10-ml aliquots of 2 M ammonium formate, 0.1 M formic acid. Radioactivity was assessed from the Cerenkov radiation. Culture and Incubation of AR4-2J Cells-AR4-2J cells were cultured in 35-mm wells in plastic culture dishes as described previously (3) . Cells were labeled with 100 µCi/ml [H]inositol for 96 h. The culture medium was changed (and fresh [H]inositol was added) at 48 h. One h before each experiment, the medium was removed and replaced with HEPES-buffered KREBS Ringer (pH 7.4 with NaOH) containing 11 mM glucose, but without added [H]inositol. Cultures were maintained at 37 °C, and then NaF was added as described in the figure legends. Incubations were terminated by aspiration of the incubation medium, and then cells were immediately lysed by addition of 1.2 ml of ice-cold medium containing 10 mM NaF, 5 mM NaEDTA, 1 mg/ml InsP, 0.1% (v/v) Triton X-100. Extracts were kept on ice for 15 min prior to their centrifugation to remove cell debris. The resultant supernatants were saved and applied to NENSORBcolumns to remove soluble protein as described above.

HPLC Analysis of Inositol Polyphosphates

Deproteinized samples were loaded onto a Partisphere 5-µm SAX HPLC column at a flow-rate of 1 ml/min using a WISP model 712 (Waters Associates), which injected each sample in aliquots interspersed with HPLC buffer A (1 mM NaEDTA) in a ratio of sample: buffer A of approximately 1:10. When required, fractions of 2.5 ml were collected during this loading procedure. The following gradient was then generated from a mixture of buffer A (1 mM NaEDTA, disodium salt) and buffer B (buffer A plus 1.3 M (NH)HPO (pH 3.8) with HPO) at a flow-rate of 1 ml/min (unless otherwise indicated): 0-2 min, B = 0%; 2-10 min, B increased linearly to 44%; 10-100 min, B increased linearly to 100% and was retained at this level until either 101 min (for older columns) or 110 min (new columns). Finally, B then returned to 0%. Either 1 or 2 min fractions were collected. A slightly different gradient was used to resolve inositol polyphosphates in extracts of intact cells: 0-10 min, B = 0%; 10-30 min, B increased linearly to 40%; 30 to 130 min, B increased linearly to 75%; 130-131, B increased linearly to 100%; 131-157 B = 100%; 157 min, B returned to 0%. One-ml fractions were collected. All HPLC fractions were mixed with 4 volumes of Monoflow 4 scintillant (National Diagnostics, Manville, NJ), and radioactivity was quantified by liquid scintillation spectrometry.


RESULTS

Phosphorylation of InsP to PP-InsP-PP by Liver Homogenate

It was recently shown that extracts of both Dictyostelium, and some mammalian cultured cell types, can phosphorylate InsP to PP-InsP(3, 4) . The initial goal of our current study was to identify a mammalian tissue that could be a useful in vitro model for the study of InsP phosphorylation. We found that liver homogenate readily converted InsP ( peak A, Fig. 1, upper panel) to PP-InsP ( peak B, Fig. 1, upper panel) when 10 mM NaF was employed to inhibit PP-InsP phosphatase ( (3) , and see below). A novel observation in our present experiments was the phosphorylation of [H]InsP to substantial amounts of an additional H-labeled product which was more polar than PP-InsP. Approximately 25% of InsP/h/(mg protein) was converted to peak C (in Fig. 1, upper panel), greatly exceeding the tiny amounts observed in some earlier experiments with extracts of cultured cells (3, 4) . We have never observed any further products which were more polar than peak C, even when the HPLC column was extensively washed with 1.5 M of [NH]HPO for 30 min (data not shown).


Figure 1: Phosphorylation of InsP and PP-InsP by liver homogenate. Liver homogenates were incubated at a final concentration of 0.9 mg protein/ml for 40 min in 0.5 ml of assay buffer 1 (see ``Experimental Procedures'') that contained either 10,000 dpm [H]InsP ( peak A, upper panel) or 5,000 dpm PP-[H]InsP ( peak B, lower panel). Reactions were quenched and extracted as described under ``Experimental Procedures,'' spiked with 600 dpm [-P]PP-InsP-PP, and analyzed by HPLC as described under ``Experimental Procedures''; one-min fractions were collected. The circles depict H dpm/fraction, and triangles depict P dpm/fraction for the internal standard. The data are representative of five experiments ( upper panel) or seven experiments ( lower panel). The difference in absolute elution times of the HPLC runs described in the upper and lower panels arises from differences in the age of the column; this is compensated for by the internal standard of [-P]PP-InsP-PP.



A likely structure for peak C is a bis-diphosphoinositol tetrakisphosphate, PP-InsP-PP (see below). However, two different structures for the material in peak C were considered: 1) a second isomer of PP-InsP, formed either by phosphorylation of InsP or by isomerization of the PP-InsP in peak B; 2) a PPP-InsP, i.e. a triphosphoinositol pentakisphosphate. Both of these alternative structures were ultimately excluded, on the basis of the data described below. Indeed, one informative experiment was the determination of the immediate precursor of the putative PP-InsP-PP. Here, PP-InsP was incubated with liver homogenate in the presence of ATP and NaF ( Fig. 1 lower panel). Little PP-InsP was dephosphorylated during 1-h incubations; instead, about 50% of the PP-InsP was itself converted to PP-InsP-PP (Fig. 1, lower panel). In other words, PP-InsP, and not InsP, is the immediate precursor of PP-InsP-PP. Indeed, the [-P]PP-InsP-PP supplied by DuPont NEN is produced from [-P]PP-InsP in an ATP-dependent enzymatic reaction, and when this material was used as an internal standard during HPLC, it co-eluted with peak C (Fig. 1).

A Substrate Cycle for InsP and PP-InsP in Liver Homogenate

In the absence of ATP and NaF, PP-InsP was rapidly dephosphorylated by liver homogenate, and no PP-InsP-PP was formed (compare Fig. 1, lower panel, with Fig. 2). We determined which phosphate group(s) was hydrolyzed by HPLC analysis of the products formed from the metabolism of a mixture of PP-[H]InsP and [-P]PP-InsP. The initial H-labeled product did not contain any P-label (Fig. 2); thus, the only significant route of dephosphorylation of PP-InsP was to InsP by removal of the -phosphate in the diphosphate group, all of which was recovered as [P]P (Fig. 2). This phosphatase activity completes the substrate cycle between InsP and PP-InsP. Note that the extract from each assay was loaded onto the HPLC in a relatively large volume (see ``Experimental Procedures''); much of the [P]P did not bind to the HPLC column, and instead was eluted in the load ( inset to Fig. 2 ).


Figure 2: Dephosphorylation of PP-InsP by liver homogenate. Liver homogenates were incubated at a final concentration of 3.5 µg protein/ml for 30 min in 1 ml of assay buffer 2 (see ``Experimental Procedures'') that contained 2500 dpm PP-[H]InsP and 1500 dpm [-P]PP-InsP. Reactions were quenched, extracted, and analyzed by HPLC as described under ``Experimental Procedures.'' 5-min fractions were collected while the sample was loaded onto the HPLC (see inset), and 2-min fractions were collected during the gradient. Closed circles depict H dpm/fraction, and open circles depict P dpm/fraction. The arrow indicates the elution position of PP-InsP-PP (determined in a separate HPLC run). The data are representative of four experiments.



Diphosphoinositol Polyphosphates Are Hydrolyzed by MIPP

Mammalian tissues contain a MIPP that has particularly high affinity toward Ins (1, 3, 4, 5, 6) P and InsP(12, 13) . We have now discovered that MIPP also dephosphorylated PP-InsP (Fig. 3): in these experiments MIPP was incubated with PP-[H]InsP and [-P]PP-InsP. The immediate products formed were [H]InsP and [P]P; no [-P]PP-InsP was detected (Fig. 3). Thus, MIPP was highly specific in its expression of monoesterase activity toward the diphosphate group in PP-InsP. This was an unexpected observation, because MIPP nonspecifically removes every phosphate from InsP, and then further hydrolyzes several of the InsP isomers that are formed (12) . Clearly, the addition of a single diphosphate group to InsP has a dramatic effect upon the specificity of this intriguing enzyme. Therefore, we next investigated if MIPP could also hydrolyze PP-InsP-PP.


Figure 3: PP-InsP metabolism by MIPP. MIPP was incubated at a final concentration of 0.02 µg protein/ml for 35 min in 0.5 ml of assay buffer 2 (see ``Experimental Procedures'') that contained 5000 dpm PP-[H]InsP and 3000 dpm [-P]PP-InsP. Reactions were quenched, extracted, and analyzed by HPLC as described under ``Experimental Procedures''; one-min fractions were collected. The closed circles depict H dpm/fraction, and the open circles depict P dpm/fraction. The data are representative of seven experiments.



MIPP was found to slowly dephosphorylate a mixture of [-P]PP-InsP-PP and PP-[H]InsP-PP (Fig. 4). The immediate H-labeled reaction product was designated as InsP-PP, since it had lost the P-label (Fig. 4), which was recovered as [P]P (data not shown). The designation of InsP-PP also has the advantage of emphasizing that it is a different isomer from PP-InsP. Indeed, upon HPLC, the peak fraction of [H]InsP-PP eluted prior to [-P]PP-InsP (Fig. 5). Thus, MIPP attacks the same diphosphate group of both PP-InsP and PP-InsP-PP. It therefore became important to determine the extent to which MIPP is responsible for the metabolism of diphosphoinositol derivatives both in vitro and in vivo (see below).


Figure 4: PP-InsP-PP metabolism by MIPP. MIPP was incubated at a final concentration of 1.5 µg protein/ml for 120 min in 1 ml of assay buffer 2 (see ``Experimental Procedures'') that contained 2500 dpm PP-[H]InsP-PP and 750 dpm [-P]PP-InsP-PP. Reactions were quenched, extracted, and analyzed by HPLC as described under ``Experimental Procedures''; one-min fractions were collected. The closed circles depict H dpm/fraction (no other dpm were present in the remainder of the gradient). The open circles depict P dpm/fraction. The data are representative of three experiments.




Figure 5: Separation of [-P]PP-InsP from [H]InsP-PP by HPLC. PP-[H]InsP-PP was dephosphorylated by MIPP to [H]InsP-PP as described in the legend to Fig. 4. The [H]InsP-PP was desalted (3) and 600 dpm were mixed with 500 dpm of an internal standard of [-P]PP-InsP which was then analyzed by HPLC as described under ``Experimental Procedures.'' Fraction size was 2 min. Closed circles depict H dpm/fraction, and triangles depict P dpm/fraction. The data are representative of seven experiments.



These data are also useful in that they confirm the actual structure of the [-P]PP-InsP-PP: recall that this is synthesized by phosphorylation of [-P]PP-InsP using non-radiolabeled ATP (see above). Therefore, an alternative triphosphate structure ( i.e. [-P]PPP-InsP) is eliminated because the P-labeled phosphate could not then have been removed by the monoesterase activity of MIPP. Furthermore, the demonstration that InsP-PP (rather than InsP) is formed by MIPP-catalyzed dephosphorylation of PP-InsP-PP, proves that the latter is indeed a more highly phosphorylated derivative of PP-InsP, rather than another isomer of PP-InsP.

Dephosphorylation of PP-InsP-PP by Liver Homogenate

The pathway of PP-InsP-PP metabolism has not previously been studied in any cell type. To address this question, liver homogenate was incubated with a mixture of [-P]PP-InsP-PP and PP-[H]InsP-PP ( peak C in Fig. 6 ); reaction products were analyzed by HPLC; there were two major H-labeled downstream metabolites, peaks D and E (Fig. 6). In five experiments (including that described by Fig. 6), the value of the [P]/[H] ratio of peak D was 93 ± 2% of the value of the [P]/[H] ratio for the PP-InsP-PP (Fig. 6). Thus, in liver homogenate the initial step in the dephosphorylation of [-P]PP-InsP-PP was near exclusively by removal of an unlabeled phosphate. Thus, peak D was either PP-InsP or PP-InsP-PP, depending upon whether a monophosphate or the unlabeled diphosphate group of [-P]PP-InsP-PP was hydrolyzed. Peak E (Fig. 6) did not contain any P-label. Therefore, the dephosphorylation of peak D to peak E occurred by specific removal of the P-labeled phosphate. Some PP-InsP-PP could also have been directly converted to peak E. In other words, three catabolic pathways were possible: 1) PP-InsP-PP PP-InsP InsP; 2) PP-InsP-PP PP-InsP-PP InsP-PP; 3) PP-InsP-PP InsP-PP.


Figure 6: PP-InsP-PP metabolism by liver homogenate. Aliquots of liver homogenate were incubated at a final concentration of 0.8 mg protein/ml for 60 min in 0.5 ml of assay buffer 2 (see ``Experimental Procedures'') containing 5000 dpm PP-[H]InsP-PP and 4000 dpm [-P]PP-InsP-PP. Reactions were quenched, extracted, and analyzed by HPLC as described under ``Experimental Procedures.'' One-min fractions were collected. Closed circles depict H dpm/fraction, and open circles depict P dpm/fraction. The data are representative of five experiments.



To help distinguish between these alternative metabolic pathways, we prepared individual, desalted (3) samples of peaks D and E that were only H-labeled ( i.e. by dephosphorylation of PP-[H]InsP-PP). Upon HPLC, peak D coeluted with a PP-InsP standard (Fig. 7, upper panel), and peak E coeluted with InsP (Fig. 7, lower panel). These data are consistent with PP-InsP-PP being metabolized to PP-InsP and then to InsP ( i.e. the first of the three options described above). Nevertheless, we sought an additional means of discriminating between the three alternative pathways. Note that two of them (options 2 and 3) predict that peak E would contain a diphosphoinositol tetrakisphosphate (InsP-PP). Only option 1 predicts that peak E is InsP. This distinction is important because we have previously demonstrated (3) that a tobacco ``pyrophosphatase'' (17) will attack diphosphoinositols but not monophosphoinositols such as InsP. We therefore incubated 800 dpm of peak E with ``pyrophosphatase'' (see ``Experimental Procedures''). In control experiments, pyrophosphatase was also incubated with 2000 dpm of H-labeled standards of both InsP and a diphosphoinositol tetrakisphosphate. Up to 77% of the latter was dephosphorylated by the pyrophosphatase in four experiments. In contrast, no detectable hydrolysis of InsP was ever observed and, importantly, pyrophosphatase did not hydrolyze peak E (data not shown). Thus, peak E contained only InsP, so peak D must be PP-InsP, itself formed by dephosphorylation of PP-InsP-PP. This pathway represents a complete reversal of the route of PP-InsP-PP synthesis, establishing two coupled substrate cycles (Fig. 8). Note also the different positional specificities of the phosphatase activities toward PP-InsP-PP as compared to PP-InsP; this may reflect the participation of two distinct enzymes.


Figure 7: HPLC analysis of the products of PP-InsP-PP metabolism. H-Labeled peaks Dand Ewere prepared by dephosphorylation of PP-[H]InsP-PP (see text and Fig. 6). In the upper panel, 600 dpm of peak D( circles) was mixed with 600 dpm of [-P]PP-InsP ( triangles) and analyzed by HPLC (see ``Experimental Procedures''). One-min fractions were collected. The data are representative of three experiments. In the lower panel, 750 dpm of peak E ( circles) were mixed with 2000 dpm [-P]PP-InsP ( triangles, peak at 74 min) and analyzed by HPLC (as described under ``Experimental Procedures,'' except that buffer B increased from 44 to 90% between 10 and 120 min). One-min fractions were collected. Immediately before the latter HPLC run, the elution positions were determined for standards of diphosphoinositol tetrakisphosphate and InsP ( triangles, peaks at 46 and 56 min, respectively); the standards were themselves spiked with [-P]PP-InsP (not shown, peak at 74 min; therefore, the column did not significantly differ in its elution characteristics between the two runs). Data are typical of four experiments.




Figure 8: Interconversion of InsP, PP-InsP and PP-InsP-PP.



MIPP selectively removed the P-labeled phosphate from [-P]PP-InsP-PP, but this reaction was not detected in experiments with liver homogenate (see above). Further evidence that MIPP cannot have made a significant contribution to the metabolism of PP-InsP-PP in vitro arose when liver homogenates were fractionated into 100,000 g particulate and soluble fractions; it was the latter which contained the bulk (70-90%) of total phosphatase activities toward PP-InsP and PP-InsP-PP (), whereas MIPP is predominantly associated with 100,000 g particulate fractions (12, 13) . Another discerning feature of PP-InsP and PP-InsP-PP dephosphorylation by MIPP was its relative insensitivity to F: in a typical experiment, the rate of metabolism of PP-InsP by MIPP (% dephosphorylation/ng protein/min) was 0.66 ± 0.04 in the absence of F and 0.55 ± 0.02 in the presence of 10 mM F ( n = 3). Similarly, the rate of metabolism of PP-InsP-PP was 0.014 ± 0.001 in the absence of F and 0.018 ± 0.001 in the presence of 10 mM F ( n = 4). In complete contrast, F strongly inhibited dephosphorylation by homogenate of both PP-InsP and PP-InsP-PP (IC 0.03 mM and 1.4 mM respectively, Fig. 9 ). It is worth emphasizing the 40-fold difference in the sensitivities of the PP-InsP phosphatase and the PP-InsP-PP phosphatase to inhibition by F. These data strengthen the idea that distinct enzymes perform these two reactions.


Figure 9: Effect of F upon metabolism of PP-InsP and PP-InsP-PP by liver homogenate. The metabolism of trace amounts of substrate was assayed as described under ``Experimental Procedures'' using assay buffer 2 with the indicated concentration of NaF. [-P]PP-InsP (1 nM) was incubated with liver homogenate (4 µg protein/ml), and metabolism was followed by assaying [P]-P release on gravity-fed columns (circles). PP-[H]InsP-PP (0.2 nM) was incubated with liver homogenate (80 µg protein/ml) and metabolism was followed by HPLC ( squares). Data are representative of two experiments.



Diphosphoinositol Polyphosphates in Intact Cells

Levels of PP-[H]InsP in intact cells are about 2-3% those of [H]InsP (Fig. 10, and see Refs. 3, 4, 9). In several earlier experiments PP-InsP-PP was not detected, possibly because of difficulties in recovering this material from cell extracts (3, 9) . Our improved methods (see ``Experimental Procedures'') now reproducibly show (Fig. 10) that AR4-2J cells contain small quantities of PP-[H]InsP-PP (0.6% of the [H]InsP peak). This compound was identified by its co-elution with a P-labeled standard (Fig. 10) and its metabolism by AR4-2J cell homogenates to compounds which, upon HPLC, were identified as PP-InsP and InsP (data not shown). Another new observation was that the PP-[H]InsP isolated from intact cells co-eluted with a [-P]PP-InsP standard (Fig. 10). Thus, in AR4-2J cells, there was no indication of the presence of significant amounts of [H]InsP-PP, which elutes 2 min prior to PP-InsP (Figs. 5 and 10).


Figure 10: Levels of [H]InsP, PP-[H]InsP, and PP-[H]InsP-PP in [H]inositol-labeled AR4-2J cells and the effect of NaF. AR4-2J cells were cultured and labeled with [H]inositol as described under ``Experimental Procedures.'' Cells were incubated for 40 min either without NaF ( panel A) or with 0.8 mM NaF ( panel B) or with 10 mM NaF ( panel C). Cells were then quenched, extracted, spiked with standards of [-P]PP-InsP plus [-P]PP-InsP-PP, and analyzed by HPLC as described under ``Experimental Procedures.'' Note the difference in scale between the x axes depicting H dpm/fraction ( circles) and P dpm/fraction ( triangles). The arrows in panel A depicts the individual peak elution positions of a mixture of standards of [H]InsP-PP and [-P]PP-InsP, determined by HPLC immediately before the sample was analyzed. The sizes of the peaks (in dpm) were as follows (from left to right): panel A, InsP = 64,729; PP-InsP = 1958; PP-InsP-PP= 402. Total [H]InsP (not shown) was 13,268. Panel B, InsP = 31,657; PP-InsP = 32244; PP-InsP-PP= 1947. Total [H]InsP (not shown) was 14,925. Panel C, InsP = 42260; PP-InsP = 24788; PP-InsP-PP= 451. Total [H]InsP (not shown) was 53,686. Data are representative of three experiments.



We have previously shown that the ability of F to inhibit PP-InsP dephosphorylation may be exploited as a metabolic trap to expose the ongoing rate of turnover of PP-InsP and InsP(3) . We have now compared the effects of F upon both PP-InsP and PP-InsP-PP. AR4-2J cells were incubated for 40 min with 0.8 mM F, which is below the level that stimulates phospholipase C through an AlF-dependent activation of GTP-binding proteins (3) . Indeed, [H]InsP levels were not significantly affected by this protocol (see legend to Fig. 10). On the other hand, Fmediated inhibition of PP-InsP phosphatase caused levels of PP-[H]InsP to increase 16-fold, at the expense of a 50% decrease in the amount of [H]InsP (Fig. 10). Presumably because of the differential sensitivities of the PP-InsP phosphatase and the PP-InsP-PP phosphatase to inhibition by F (Fig. 9), the anion only elevated levels of PP-[H]InsP-PP 5-fold.

In cells treated with 10 mM F, phospholipase C was activated and total [H]InsP increased 4-fold (legend to Fig. 10 ). Levels of PP-[H]InsP were still greatly elevated (13-fold), but levels of PP-[H]InsP-PP were then similar to those of control cells (Fig. 10). Thus, increasing [F] from 0.8 to 10 mM had disparate effects upon the amounts of these two diphosphoinositol polyphosphates in intact cells, indicating that there are some important quantitative and perhaps qualitative differences in the processes that control their turnover.


DISCUSSION

We have established that PP-InsP-PP is formed by a mammalian tissue; we have also delineated the pathway by which PP-InsP-PP is synthesized and metabolized in vitro and in vivo. This novel information was obtained with the aid of newly available H- and P-labeled substrates, the metabolism of which was analyzed by anion-exchange HPLC. We also utilized two purified enzyme preparations (MIPP (13) and tobacco pyrophosphatase (17) ) in structurally diagnostic experiments. A fundamental conclusion of our study is that two coupled substrate cycles participate in the interconversion of PP-InsP-PP and InsP (Fig. 8). It will be important to determine how these reactions are regulated, since there are many precedents for the activities of substrate cycles being targets of intracellular control processes. Indeed, the importance of the substrate cycles that closely control the levels of individual inositol lipids are well recognized to be key elements in the regulation of cell-signaling itself. Thus, our new data represent a solid foundation for further exploration into the mechanisms by which the metabolism of PP-InsP and PP-InsP-PP might be regulated and what the physiological significance of these compounds might be.

In [H]inositol-labeled AR4-2J cells, only small levels of PP-[H]InsP and PP-[H]InsP-PP were detected (Fig. 10), even in comparison with total [H]InsP (1 µM(18) ). However, the absolute levels of both diphosphoinositols may not be fully represented by the data in Fig. 10because InsP, with which these compounds are in isotopic equilibrium (3, 9) , is not itself completely labeled in our studies (see Ref. 18). In any case, it is the rate of turnover of PP-InsP and PP-InsP-PP that is so intriguing. Here, F is a useful analytical tool; this anion strongly inhibited PP-InsP phosphatase ( Fig. 9and Ref. 3), exposing the ongoing rate of PP-InsP synthesis (Fig. 10). PP-InsP-PP phosphatase is inhibited less potently by F (Fig. 9), which may at least partly explain why treatment of intact cells with 0.8 mM F only elevated levels of PP-InsP-PP 5-fold, well below the 16-fold increase in levels of PP-InsP (Fig. 10). Thus, we are very likely to be underestimating the true extent of PP-InsP-PP turnover in intact cells. Despite this, it was an important observation that further increasing the [F] to 10 mM prevented the rise in PP-InsP-PP levels from being ob-served (Fig. 10). In contrast, the levels of PP-InsP were similar when cells were treated with either 0.8 or 10 mM F (Fig. 10). Experiments with cell-free systems showed that the PP-InsP kinase was not directly inhibited by 10 mM F (data not shown). However, F could have acted on the kinase indirectly, perhaps secondarily to the activation of phospholipase C. Indeed, Ca mobilization has already been reported to inhibit the InsP kinase (9) , and it is possible that the PP-InsP kinase is a more sensitive target. Further studies into this aspect of the regulation of diphosphoinositol polyphosphate metabolism will be an important future direction, aided by our characterization of the pathway of PP-InsP-PP synthesis and metabolism.

The differential sensitivity to F of PP-InsP phosphatase and PP-InsP-PP phosphatase (Fig. 9) implies that these two dephosphorylations may be performed by distinct enzymes. This possibility is consistent with the different positional specificity of the two reactions, which, incidentally, contrasts with the reactivity of MIPP toward the same diphosphate group on both compounds. The purification of the phosphatases that participate in the metabolism of the diphosphoinositols will provide more definite information on their number and their specificity.

Vesicle trafficking seems likely to be one area of cell biology that may be relevant to the function of diphosphoinositol polyphosphates. AP-2, AP-3, and coatomer all help regulate different aspects of vesicle traffic (7, 8) . These proteins share the ability to bind PP-InsP tighter than any other known ligand (5, 6) . In the case of coatomer, binding of PP-InsP was associated with gating of the inherant K-channel activity of this protein complex (6) . PP-InsP binding to AP-3 inhibited this protein's ability to promote clathrin assembly (5) . Our understanding of the structural specificity of both ligand binding and the ensuing physiological effects would now be improved if we investigated the interactions of PP-InsP-PP with these proteins and the cellular processes that they regulate. The relative cellular concentrations of PP-InsP and PP-InsP-PP (described in Fig. 10 ) will also help evaluate the physiological significance of any effects of these polyphosphates that we observe in vitro.

MIPP, which attacks Ins (1, 3, 4, 5) P, Ins (1, 3, 4, 5, 6) P, and InsP(12) , has now been shown to specifically attack the same diphosphate group of PP-InsP and PP-InsP-PP (Figs. 3 and 4). Since MIPP is nonspecific in its removal of every phosphate of InsP, our data demonstrate that the diphosphate groups confer a profound increase in the specificity of this enzyme. This will likely prove to be a significant observation in the elucidation of the tertiary structure of the active site. On the other hand, experiments with both homogenates and intact cells indicated that MIPP did not significantly contribute to the metabolism of either PP-InsP-PP or PP-InsP. As it happens, MIPP resides inside the endoplasmic reticulum (at least in liver) (19) . We have found that both PP-InsP and PP-InsP-PP are unable to gain access to MIPP in hepatic microsomes unless they are permeabilized with detergent (data not shown). This segregation of MIPP from its potential substrates probably explains why intact AR4-2J cells do not contain significant levels of InsP-PP, which is the unique product of MIPP-dependent attack on PP-InsP-PP. However, the mammalian erythrocyte is an example of a cell that contains MIPP in its plasma membranes (13) . Perhaps MIPP has greater access to diphosphoinositol polyphosphates in this and maybe some other cell types; our data show that one investigative approach to this issue would be to search for InsP-PP in intact cells.

In conclusion, our characterization of the pathway of synthesis and metabolism of PP-InsP-PP establishes a framework for further studies into the physiological significance of diphosphoinositol polyphosphates. We can surely be confident that the cell reaps a substantial reward for investing considerable amounts of cellular energy into the ongoing turnover of these compounds.

  
Table: Phosphatase activities towards PP-InsP and PP-InsP-PP in the 100,000 g supernatant and particulate fractions of liver homogenate

Homogenates, and 100,000 g soluble and particulate fractions, were prepared and assayed for phosphatase activities as described under ``Experimental Procedures'' using 4 µg protein ml (for PP-InsP phosphatase) or 40 µg protein/ml (for PP-InsP-PP phosphatase). PP-InsP phosphatase activity was assayed by measuring [P]-P release from [P]PP-InsP using gravity fed columns. Dephosphorylation of PP-[H]InsP-PP was assayed by HPLC. Reaction rates were calculated using the first-order rate equation, i.e. [ S]= [ S] e, where k is the first-order rate constant in units of min, and [ S] is the concentration of substrate at times 0 and t. Data are means ± standard errors from the number of experiments indicated in parentheses.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 919-541-0793; Fax: 919-541-1898.

The abbreviations used for the inositol phosphates follow IUPAC nomenclature ( i.e. Ins(1,4,5)P represents inositol 1,4,5-triphosphate); InsP-PP, diphosphoinositol tetrakisphosphate (a diesterphosphate derivative of InsP); PP-InsP (formerly InsPP (3)) and InsP-PP, diphosphoinositol pentakisphosphate isomers (diesterphosphate derivatives of InsP) which differ in the position of the diphosphate group on the inositol ring; PP-InsP-PP, bis-diphosphoinositol tetrakisphosphate. MIPP, multiple inositol polyphosphate phosphatase (formerly Ins(1,3,4,5)P 3-phosphatase (see Ref. 13)); HPLC, high performance liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; dpm, disintegrations/min.

B. Fleischer, S. Fleischer, and S. B. Shears, unpublished data.


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