©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel, Phospholipase C-independent Pathway of Inositol 1,4,5-Trisphosphate Formation in Dictyostelium and Rat Liver (*)

(Received for publication, July 13, 1995; and in revised form, October 6, 1995)

Peter Van Dijken (1) Jan-Roelof de Haas (1) Andrew Craxton (2) Christophe Erneux (3) Stephen B. Shears (2) Peter J. M. Van Haastert (1)(§)

From the  (1)Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands, (2)Inositol Lipid Section, Laboratory of Cellular and Molecular Pharmacology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, and (3)Institut de Recherche Interdisciplinaire (IRIBHN), Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium 1070

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In an earlier study a mutant Dictyostelium cell-line (plc) was constructed in which all phospholipase C activity was disrupted and nonfunctional, yet these cells had nearly normal Ins(1,4,5)P(3) levels (Drayer, A. L., Van Der Kaay, J., Mayr, G. W, Van Haastert, P. J. M. (1990) EMBO J. 13, 1601-1609). We have now investigated if these cells have a phospholipase C-independent de novo pathway of Ins(1,4,5)P(3) synthesis. We found that homogenates of plc cells produce Ins(1,4,5)P(3) from endogenous precursors. The enzyme activities that performed these reactions were located in the particulate cell fraction, whereas the endogenous substrate was soluble and could be degraded by phytase. We tested various potential inositol polyphosphate precursors and found that the most efficient were Ins(1,3,4,5,6)P(5), Ins(1,3,4,5)P(4), and Ins(1,4,5,6)P(4). The utilization of Ins(1,3,4,5,6)P(5), which can be formed independently of phospholipase C by direct phosphorylation of inositol (Stephens, L. R. and Irvine, R. F.(1990) Nature 346, 580-582), provides Dictyostelium with an alternative and novel pathway of de novo Ins(1,4,5)P(3) synthesis. We further discovered that Ins(1,3,4,5,6)P(5) was converted to Ins(1,4,5)P(3) via both Ins(1,3,4,5)P(4) and Ins(1,4,5,6)P(4). In the absence of calcium no Ins(1,4,5)P(3) formation could be observed; half-maximal activity was observed at low micromolar calcium concentrations. These reaction steps could also be performed by a single enzyme purified from rat liver, namely, the multiple inositol polyphosphate phosphatase. These data indicate that organisms as diverse as rat and Dictyostelium possess enzyme activities capable of synthesizing the second messengers Ins(1,4,5)P(3) and Ins(1, 3, 4, 5)P(4) via a novel phospholipase C-independent pathway.


INTRODUCTION

It is now well established that the occupation of cell-surface receptors on many mammalian cells leads to an activation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (^1)hydrolysis by phospholipase C (PLC), (^2)and the concomitant release of inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)), which mobilizes cellular Ca stores (Berridge and Irvine, 1989). Termination of the signaling activities of Ins(1,4,5)P(3) is accomplished by a 5-phosphatase (reviewed by Verjans et al.(1994)) to form Ins(1,4)P(2). In addition an Ins(1,4,5)P(3) 3-kinase (reviewed by Shears(1992)) yields the putative second messenger Ins(1,3,4,5)P(4) (see Irvine(1992)). Several aspects of this signal transduction process are present in the cellular slime mold Dictyostelium discoideum, which, due to its genetic tractability, is therefore a useful model system. For example, Dictyostelium cells possess cell-surface receptors for the chemoattractant cAMP that are coupled via G-proteins to a phospholipase C (Bominaar and Van Haastert, 1994); thus, extracellular stimulation of cells with cAMP elevates intracellular Ins(1,4,5)P(3) levels (Van Haastert, 1989). There is also evidence that in Dictyostelium Ins(1,4,5)P(3) releases Ca from nonmitochondrial stores (Europe-Finner and Newell, 1986; Flaadt et al., 1993).

The Dictyostelium gene for phospholipase C has been cloned, and the enzyme appeared to be structurally similar to PLC- found in mammalian cells (Drayer and Van Haastert, 1992). Subsequently, a Dictyostelium cell line with a disrupted phospholipase C gene was constructed (Drayer et al., 1994). These plc cells had no measurable phospholipase C activity, but they expressed a normal phenotype. More surprisingly, the basal levels of Ins(1,4,5)P(3) were only 20% lower than that of wild-type cells (Drayer et al., 1994). This observation led to the suggestion that there might be a phospholipase C-independent source of Ins(1,4,5)P(3). This suggestion is without precedent, and even the demonstration that Dictyostelium can convert Ins(1,3,4,5)P(4) to Ins(1,4,5)P(3) by a 3-phosphatase activity (Van Dijken et al., 1995) does not resolve this problem, since the only known source for Ins(1,3,4,5)P(4) is Ins(1,4,5)P(3) itself.

These results raise fundamental questions concerning the sources of both Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4) in both wild-type and plcDictyostelium cells. We now describe a novel metabolic pathway by which these two inositol phosphates can be formed by dephosphorylation of Ins(1,3,4,5,6)P(5). Moreover, we further discovered that these enzyme activities were similar to those that can also be catalyzed by a single mammalian enzyme that was recently renamed as a multiple inositol polyphosphate phosphatase (MIPP) (Craxton et al., 1995). These observations indicate that we may have to reevaluate the mechanisms by which cellular Ins(1,4,5)P(3) is generated and utilized.


EXPERIMENTAL PROCEDURES

Materials

InsP(6), phytase from Aspergillus ficuum and 2,3-diphospho-D-glycerate were from Sigma; Ins(1,3,4,5,6)P(5) and Ins(1,3,4,5)P(4) were from Boehringer. Nuclepore filters were from Costar. [^3H]Ins(1,3,4,5)P(4) (21 Ci/mmol) and [^3H]InsP(6) (24.4 Ci/mmol) were from DuPont NEN. [^3H]Ins(1,4,5)P(3) (50 Ci/mmol) was from Amersham Corp. The Zorbax SAX HPLC column was from DuPont. Quard column resin was obtained from Chrompack. Type I Ins(1,4,5)P(3) 5-phosphatase was purified (four purification steps) from bovine brain to a specific activity of 5 µmolbulletminbulletmg. Recombinant rat brain Ins(1,4,5)P(3) 3-kinase (clone C5 in Takazawa et al.(1990)) was expressed and purified as described by Takazawa and Erneux(1991) to a specific activity of approximately 1 µmolbulletminbulletmg. The preparation was free of contaminating ATPase activity.

[3-P]Ins(1,3,4,5)P(4) was prepared from Ins(1,4,5)P(3) and [-P]ATP as described in Van Dijken et al.(1994). [^3H]Ins(1,3,4,5,6)P(5) and DL-[^14C]Ins(1,4,5,6)P(4) were isolated, respectively, from [^3H]inositol-labeled or [^14C]inositol-labeled turkey erythrocytes (Stephens and Downes, 1990). [^3H]Ins(1,4,5,6)P(4) was synthesized as described by Craxton et al.(1994). Ins(1,2,4,5,6)P(5) was prepared as described by Ye et al.(1995).

Preparation of Ins(1,2,3,4,5)P(5)

Ins(1,2,3,4,5)P(5) was prepared from InsP(6) using phytase isolated from Paramecium essentially as described in Freund et al.(1992) and modified by Van Der Kaay and Van Haastert (^3)a 100-µl incubation containing 10 mM Tris, pH 7.0, 1 mM InsP(6), 15,000 dpm [^3H]InsP(6), and 20 µl of isolated phytase was incubated for 1 h at room temperature. Reactions were terminated by boiling for 5 min, after which the sample was applied to an HPLC column (see below). The fractions containing [^3H]InsP(5) were collected and desalted by dialysis (Van Der Kaay and Van Haastert, 1995). The product was shown to be D-Ins(1,2,3,4,5)P(5) by Van Der Kaay and Van Haastert.^3 The radioactivity in the sample was used to calculate the concentration of Ins(1,2,3,4,5)P(5).

Preparation of Ins(1,4,5,6)P(4)

Ins(1,4,5,6)P(4) was prepared by incubation for 30 min at room temperature of a 250-µl incubation containing 20 mM triethanolamine, pH 6.5, 5.9 mM EGTA, 0.5 mM EDTA, 10 mM CaCl(2), 3500 dpm [^3H]Ins(1,3,4,5,6)P(5), 100 µM Ins(1,3,4,5,6)P(5), and 100 µl of Dictyostelium homogenate (see below). The reaction was stopped by boiling and analyzed by HPLC (see below). The second [^3H]InsP(4) peak (Ins(1,4,5,6)P(4); see ``Results'') was isolated using HPLC. After dialysis for 2 h (Van Der Kaay and Van Haastert, 1995), the sample was freeze-dried and redissolved in distilled water. The radioactivity in the sample was used to calculate the concentration of Ins(1,4,5,6)P(4).

Growth of Dictyostelium Cells and Preparation of Subcellular Fractions

Dictyostelium plc (HD10) cells were grown as described in Drayer et al.(1994). Cells were collected by centrifugation (3 min, 300 times g). After washing twice in TEE buffer (20 mM triethanolamine, pH 6.5, 5.9 mM EGTA, 0.5 mM EDTA), cells were lysed at 10^8 cells/ml in TEE buffer by pressing them through a Nuclepore filter (pore size 3 µm) (Das and Henderson, 1983). Soluble and particulate cell fractions were obtained after centrifugation of the homogenate at 140,000 times g for 40 min. The pellet was resuspended in the original volume of TEE buffer. Boiled particulate and soluble fraction were prepared by boiling the sample for 5 min.

A phytase-treated soluble fraction was prepared by overnight incubation of 250 µl of soluble fraction in a 285-µl incubation with 0.2 units/ml A. ficuum phytase at 55 °C in the presence of 50 mM sodium acetate, pH 5.15. The reaction was stopped by boiling for 60 min to totally inactivate the phytase. In a control experiment, added [^3H]InsP(6) (15,000 dpm) was totally degraded by the phytase to [^3H]InsP and [^3H]inositol.

Ins(1,4,5)P(3) Formation Assay

Ins(1,4,5)P(3) formation from endogenous substrates in cell lysates of Dictyostelium was assayed by incubating either particulate or soluble fraction or a 1:1 mixture of both in a final volume of 55 µl containing the indicated concentrations of CaCl(2) and MgCl(2) at approximately 5 times 10^7 lysed cells/ml. Ins(1,4,5)P(3) formation from exogenous substrates was carried out by incubating particulate cell fraction (10^8 lysed cells/ml) with an equal volume of TEE buffer containing unlabeled inositol phosphates in the presence or absence of CaCl(2) or MgCl(2) (concentrations as indicated). Reactions were stopped after 0 and 30 min with 1 volume of 3.5% perchloric acid. The samples were neutralized by the addition of 50% saturated KHCO(3). Ins(1,4,5)P(3) cross-reactivity in the samples was determined using an isotope dilution assay as described by Van Haastert(1989).

Degradation of Radiolabeled Inositol Phosphates by Dictyostelium Particulate Fraction

Radiolabeled inositol phosphates were incubated at room temperature in 100-µl incubations containing 50 µl of particulate cell fraction (5 times 10^7 cell equivalent/ml final) in the presence of 10 mM CaCl(2) in TEE buffer for various times. The reaction was stopped by boiling for 5 min. After centrifugation (5 min at 14,000 times g) the supernatant was analyzed by HPLC (see below).

HPLC Analysis of Inositol Phosphates

Samples containing radiolabeled inositol phosphates were analyzed using a Zorbax HPLC column (6.2 mm times 8 cm) equipped with a guard column (5 mm times 10 cm). The column was eluted with a gradient of water (buffer A) and 1.2 M (NH(4))(2)HPO(4), pH 3.2 (buffer B) at 1.5 ml/min as indicated in the figures. Unless mentioned otherwise, fractions of 0.5 ml were collected, to which 4 ml of scintillation mixture (Packard) was added. Radioactivity in the fractions was determined in a liquid scintillation counter. Alternatively, an Adsorbosphere SAX column was used as described by Menniti et al.(1990).

Identification of Ins(1,4,5)P(3) Cross-reactivity Using Purified Ins(1,4,5)P(3) 3-Kinase

A sample with known Ins(1,4,5)P(3) cross-reactivity was mixed with 1000 dpm [^3H]Ins(1,4,5)P(3) and incubated at 37 °C with 6 µl of purified Ins(1,4,5)P(3) 3-kinase in a 106-µl incubation containing 100 mM Hepes, pH 7.5, 20 mM MgCl(2), 1 mM ATP, 1 mg/ml BSA. The reaction was stopped by boiling after 0 and 12 min. Of the sample, 19 µl was analyzed in triplicate for Ins(1,4,5)P(3) cross-reactivity (see above), and the remainder was analyzed by HPLC (see above) for conversion of [^3H]Ins(1,4,5)P(3) to [^3H]Ins(1,3,4,5)P(4).

Identification of Ins(1,4,5)P(3) Cross-reactivity Using Purified Ins(1,4,5)P(3)/Ins(1,3,4,5)P(4) 5-Phosphatase

A sample with known Ins(1,4,5)P(3) cross-reactivity was mixed with 1000 dpm of [^3H]Ins(1,4,5)P(3) and incubated at 37 °C with 5 µl of purified Ins(1,4,5)P(3) 5-phosphatase in a 106-µl incubation containing 50 mM Hepes, pH 7.5, 2 mM MgCl(2), 1 mg/ml BSA, 10 mM 2-mercaptoethanol. The reaction was stopped by boiling after 0 and 10 min. Of the sample, 19 µl was analyzed for Ins(1,4,5)P(3) cross-reactivity in triplicate (see above), and the remainder of the sample was analyzed by HPLC (see above) for conversion of [^3H]Ins(1,4,5)P(3) to [^3H]Ins(1,4)P(2).

Identification of [^3H]InsP(3) Using Purified Ins(1,4,5)P(3) 3-Kinase and 5-Phosphatase

A sample of HPLC-purified [^3H]InsP(3) was split into three aliquots. One aliquot was immediately analyzed by HPLC (see above). The second aliquot was incubated in 1200 µl in the presence of 100 mM Hepes, pH 7.5, 20 mM MgCl(2), 1 mM ATP, 1 mg/ml BSA with 12 µl of purified Ins(1,4,5)P(3) 3-kinase for 13 min at 37 °C. The reaction was stopped by boiling for 5 min, after which the sample was analyzed using HPLC for conversion of [^3H]InsP(3) to [^3H]Ins(1,3,4,5)P(4). The third aliquot of [^3H]InsP(3) was added to an identical control incubation except that an excess of genuine [^3H]Ins(1,4,5)P(3) was also present in order to monitor formation of authentic [^3H]Ins(1,3,4,5)P(4).

Identification with Type I Ins(1,4,5)P(3)/Ins(1,3,4,5)P(4) 5-phosphatase was carried out similarly except that the reaction mixture contained 100 mM Hepes, pH 7.5, 1 mg/ml BSA, 2 mM MgCl(2), 5 mM 2-mercaptoethanol, and the sample was incubated for 10 min. at 37 °C.

Identification of [^3H]Ins(1,4,5,6)P(4) Using Ins(1,4,5,6)P(4) 3-Kinase

[^3H]Ins(1,4,5,6)P(4) was identified by phosphorylating it with a partly purified Ins(1,4,5,6)P(4) 3-kinase (Craxton et al., 1994). Briefly, samples were incubated with purified Ins(1,4,5,6)P(4) 3-kinase for 2 h at 37 °C in 0.7 ml of buffer containing 10 mM Hepes, pH 7.2, 5 mM MgSO(4), 4 mM ATP, 9 mM phosphocreatine, 15 units of creatine phosphokinase, and 0.2 mg of BSA. Samples were quenched with perchloric acid, neutralized with freon/octylamine (Kirk et al., 1990), and spiked with approximately 700 dpm of an internal standard of DL-[^14C]Ins(1,4,5,6)P(4). Samples were chromatographed on an Adsorbosphere SAX column.


RESULTS

Ins(1,4,5)P(3) Formation in Dictyostelium Lysates from Endogenous Substrates

A Dictyostelium mutant with a disrupted PLC gene (plc cells) had no detectable phospholipase C activity. In order to account for why these plc cells contained nearly normal levels of Ins(1,4,5)P(3) (Drayer et al., 1994), we first investigated if the formation of this compound from endogenous precursors could be detected in cell free extracts. Ins(1,4,5)P(3) was initially assayed by measuring cross-reactivity with an Ins(1,4,5)P(3)-binding protein (see ``Experimental Procedures''). The extent to which any Ins(1,4,5)P(3) would accumulate in such experiments depended upon the rate of Ins(1,4,5)P(3) synthesis relative to its rate of degradation. A variety of incubation conditions were analyzed in an effort to limit Ins(1,4,5)P(3) degradation without inadvertently inhibiting its synthesis. The incubation conditions described in the legend to Fig. 1gave an optimal rate of accumulation of Ins(1,4,5)P(3) cross-reactivity. These incubations contained 10 mM calcium but no added magnesium. Under such conditions, significant amounts of Ins(1,4,5)P(3) cross-reactivity were produced from endogenous precursors in a cell-free lysate both from plc cells (Fig. 1A) and wild-type cells (data not shown).


Figure 1: Formation of Ins(1,4,5)P(3) cross-reactivity by Dictyostelium cellular fractions from endogenous and exogenous substrates. A, Dictyostelium particulate and soluble fraction were obtained by centrifugation of a lysate. Some fractions were boiled or treated with phytase before they were incubated as indicated in the figure. Samples were incubated in the presence of 10 mM CaCl(2) for 0 and 30 min and then quenched and neutralized as described under ``Experimental Procedures.'' 20-µl aliquots of the final, quenched extracts were analyzed for Ins(1,4,5)P(3) cross-reactivity by the isotope dilution assay using Ins(1,4,5)P(3) binding protein. The data in the figure were obtained by subtracting the cross-reactivity at zero time from that at 30 min. Data shown are the means and standard errors of mean of at least three experiments. Note that the samples soluble + phytase + particulate and soluble + boiled phytase + particulate (which serves as a control for the former) are 5% more diluted than the other samples. Bars indicated with a star indicate Ins(1,4,5)P(3) cross-reactivity accumulation during the incubation time, that is significantly different from zero as assessed by Student's t test (p leq 0.05). B, unlabeled inositol phosphates were incubated for 0 and 30 min with Dictyostelium particulate fraction in the presence of 10 mM CaCl(2). Ins(1,4,5)P(3) cross-reactivity accumulating per 10 pmol of substrate is shown as mean and standard error of the mean. The experiments were performed at least in triplicate. The amount of Ins(1,4,5)P(3) cross-reactivity accumulating during the incubation period is for all values significantly different from zero as assessed by Student's t test (p leq 0.05).



Ins(1,4,5)P(3)/Ins(1,3,4,5)P(4) 5-phosphatase and Ins(1,4,5)P(3) 3-kinase were used to investigate the extent to which the material that expressed Ins(1,4,5)P(3) cross-reactivity was genuine Ins(1,4,5)P(3), as follows. Samples with known amounts of Ins(1,4,5)P(3) cross-reactivity were prepared from incubations containing cell lysate as described in the legend to Fig. 1A. [^3H]Ins(1,4,5)P(3) was added to these samples, which were then incubated with either 5-phosphatase or 3-kinase. After such incubations, the residual Ins(1,4,5)P(3) cross-reactivity was determined and compared with the amount of metabolism of [^3H]Ins(1,4,5)P(3). Both with 5-phosphatase and 3-kinase, the decrease in Ins(1,4,5)P(3) cross-reactivity amounted to 75% of the amount of [^3H]Ins(1,4,5)P(3) metabolized (data not shown). Thus 75% of the material that expressed Ins(1,4,5)P(3) cross-reactivity represented authentic Ins(1,4,5)P(3). The remaining 25% of the Ins(1,4,5)P(3) cross-reactivity was not further investigated but could have been due to the presence of other inositolphosphates. For example Ins(1,2,4)P(3)/Ins(2,3,6)P(3) has previously been shown to be a potent ligand of the Ins(1,4,5)P(3) receptor (Freund et al., 1992).

The Ins(1,4,5)P(3)-forming activity present in the homogenate required both soluble and particulate components, as these separate fractions did not form Ins(1,4,5)P(3) unless mixed (Fig. 1A). The factor in the particulate fraction was heat-labile, whereas the soluble factor was heat stable. Preincubation of the soluble fraction with the inositol polyphosphate-degrading enzyme, phytase, abolished Ins(1,4,5)P(3) formation (Fig. 1A). These data suggest that the particulate cell fraction contains enzyme activities that form Ins(1,4,5)P(3), whereas the soluble fraction contains endogenous substrates that are presumably inositol polyphosphates, because they are degraded by phytase.

Ins(1,4,5)P(3) Formation in Dictyostelium Particulate Fraction from Exogenous Substrates

Various exogenous inositol polyphosphates containing phosphates at positions 1, 4, and 5 were incubated with a plc particulate fraction, and the amount of Ins(1,4,5)P(3) cross-reactivity that accumulated was determined (Fig. 1B). Ins(1,3,4,5)P(4) was the most efficient precursor of Ins(1,4,5)P(3) cross-reactivity. In addition we found that both Ins(1,4,5,6)P(4) and Ins(1,3,4,5,6)P(5) were precursors that produced approximately half as much Ins(1,4,5)P(3) cross-reactivity as that generated from Ins(1,3,4,5)P(4). Further experiments were performed to confirm that authentic Ins(1,4,5)P(3) was produced from these particular precursors (see below). InsP(6), Ins(1,2,3,4,5)P(5), and Ins(1,2,4,5,6)P(5) also generated minor amounts of Ins(1,4,5)P(3) cross-reactivity (only 1-5% as much as Ins(1,3,4,5)P(4)). Although the amounts of Ins(1,4,5)P(3) cross-reactivity formed from these compounds were statistically significant different from zero, as assessed by the student t test (p leq 0.05), the biological significance is unclear. All of these latter three compounds are potential precursors of the potent ligand of the Ins(1,4,5)P(3) receptor, Ins(1,2,4)P(3)/Ins(2,3,6)P(3) (Freund et al., 1992). The low levels of Ins(1,4,5)P(3) cross-reacting material generated from these compounds precluded further investigation into this possibility.

Ca Dependence of Ins(1,4,5)P(3) Cross-reactivity Accumulation

The Ca dependence of Ins(1,4,5)P(3) cross-reactivity accumulation from Ins(1,3,4,5,6)P(5) and Ins(1,3,4,5)P(4) was first determined in the absence of Mg (Fig. 2). The data were analyzed according to the Hill equation: V = (V(max))*[Ca]^n/([Ca]^n + [K]^n), where K is the Ca concentration at which half-maximal activity is observed, and n is the Hill coefficient. The obtained Hill coefficients were 1.2 ± 0.3 and 1.1 ± 0.2 for Ins(1,3,4,5,6)P(5) and Ins(1,3,4,5)P(4), respectively. The Ca concentrations at which half-maximal activity was observed (K) were 7.5 ± 1.9 µM and 4.1 ± 0.9 µM, respectively (n set at 1.0). These activation constants are similar for both substrates.


Figure 2: Calcium dependence of the accumulation of Ins(1,4,5)P(3) cross-reactivity. The accumulation of Ins(1,4,5)P(3) cross-reactivity was determined at various free CaCl(2) concentrations, under conditions described in Fig. 1B, with Ins(1,3,4,5)P(4) (circle) or Ins(1,3,4,5,6)P(5) (up triangle) as substrates. Ins(1,3,4,5,6)P(5) was also used as substrate () for Ins(1,4,5)P(3) in the presence of 1 mM MgCl(2) and 25 mM LiCl, and 0.5 mM 2,3-diphospho-D-glycerate as inhibitors of Ins(1,4,5)P(3) degradation. The accumulation of Ins(1,4,5)P(3) cross-reactivity is expressed as a percentage of maximal Ins(1,4,5)P(3) accumulation. The data presented in this figure were fitted with the Hill coefficient set at 1. Free Ca concentrations were set using Ca/EGTA buffers as described by Bartfai(1979).



The Ca dependence was also investigated in the presence of 1 mM MgCl(2). As MgCl(2) strongly enhances the degradation of Ins(1,4,5)P(3), two inhibitors of Ins(1,4,5)P(3) phosphatases were included: lithium to inhibit Ins(1,4,5)P(3) 1-phosphatase and 2,3-diphospho-D-glycerate to inhibit Ins(1,4,5)P(3) 5-phosphatase (Van Lookeren Campagne et al., 1988). In the absence of CaCl(2) no accumulation of Ins(1,4,5)P(3) cross-reactivity was observed. However, at submicromolar Ca concentration Ins(1,4,5)P(3) formation was detectable; half-maximal activity was observed at 1.3 µM Ca (Fig. 2).

In the remaining part of this study we use conditions in which no MgCl(2) but high concentrations of total CaCl(2) (10 mM) are present. These conditions are used as a tool to further explore the formation of Ins(1,4,5)P(3) as under these conditions Ins(1,4,5)P(3) phosphatase activity is greatly reduced, which precludes the need of phosphatase inhibitors that might interfere with the phosphatases under investigation.

Route of Ins(1,4,5)P(3) Formation from Ins(1,3,4,5,6)P(5)

The major goal of this study was to determine if in Dictyostelium there was a precursor for the de novo synthesis of Ins(1,4,5)P(3) that could be synthesized independently of phospholipase C. Thus it was striking that Ins(1,3,4,5,6)P(5) was a relatively efficient precursor of Ins(1,4,5)P(3) cross-reactivity (Fig. 1B and Fig. 2) while also being an intermediate in the stepwise phosphorylation of inositol to InsP(6) (Stephens and Irvine, 1990). We therefore studied the route of degradation of Ins(1,3,4,5,6)P(5) in more detail using radiolabeled material. [^3H]Ins(1,3,4,5,6)P(5) was incubated with a particulate fraction from plcDictyostelium cells in the presence of CaCl(2). A typical degradation profile is shown in Fig. 3A. The [^3H]InsP(3) formed from the degradation of [^3H]Ins(1,3,4,5,6)P(5) was isolated using HPLC and desalted by dialysis (Van Der Kaay and Van Haastert, 1995). Identification of the [^3H]InsP(3) with purified recombinant Ins(1,4,5)P(3) 3-kinase revealed that it contained between 51 and 56% authentic [^3H]Ins(1,4,5)P(3). In a second experiment the [^3H]InsP(3) was incubated with Ins(1,4,5)P(3)/Ins(1,3,4,5)P(4) 5-phosphatase; between 67 and 72% of the [^3H]InsP(3) was genuine [^3H]Ins(1,4,5)P(3). These results confirm that a large proportion of the Ins(1,4,5)P(3) cross-reactivity formed from Ins(1,3,4,5,6)P(5) (Fig. 1B and 2) is indeed genuine Ins(1,4,5)P(3).


Figure 3: Identification of InsP(4) isomers formed from Ins(1,3,4,5,6)P(5) degradation by Dictyostelium particulate fraction. A, [^3H]Ins(1,3,4,5,6)P(5) (about 4000 dpm) was incubated with Dictyostelium particulate fraction (5 times 10^7 lysed cells/ml) for 30 min as described under ``Experimental Procedures.'' The sample was analyzed on a Zorbax SAX column eluted with a gradient that does not separate InsP(4) isomers. The gradient used was 0 min, 0% B; 10 min, 100% B. Fractions of 0.75 ml were collected. The InsP(4) fractions obtained in an experiment similar to that described in panel A were desalted and used in the experiments presented in panels B and C. B, an aliquot of the InsP(4) fraction (see panel A) was incubated in the absence (box, 600 dpm) or presence (, 950 dpm) of Ins(1,4,5, 6)P(4) 3-kinase as described under ``Experimental Procedures,'' and chromatographed such that InsP(4) isomers were separated on an Adsorbosphere SAX column as described by Menniti et al.(1990). The second InsP(4) peak, co-eluting with the internal standard of DL-[^14C]Ins(1,4,5,6)P(4), was converted by the 3-kinase, identifying it as Ins(1,4,5,6)P(4). C, an aliquot of the InsP(4) fraction (see panel A) was incubated in the presence (, 500 dpm) or absence (box, 400 dpm) of purified Ins(1,4,5)P(3)/Ins(1,3,4,5)P(4) 5-phosphatase as described under ``Experimental Procedures'' and analyzed on a Zorbax SAX column that was eluted with a gradient such that InsP(4) isomers were separated. The gradient used was 0 min, 0% B; 5 min, 30% B; 35 min, 46% B; 36 min, 100% B; 1-ml fractions were collected. The first InsP(4) peak, co-eluting with an internal [P]Ins(1,3,4,5)P(4) standard, was degraded by the 5-phosphatase, identifying it as Ins(1,3,4,5)P(4).



The total [^3H]InsP(4) fraction formed following [^3H]Ins(1,3,4,5,6)P(5) degradation by Dictyostelium particulate fraction (see Fig. 3A) was isolated, desalted, and rechromatographed using a different HPLC system that resolved this material into two InsP(4) peaks. Fig. 3B shows that the InsP(4) fraction contained two main InsP(4) isomers. Incubation of this material with purified Ins(1,4,5,6)P(4) 3-kinase revealed that 88% of the second InsP(4) peak was converted to Ins(1,3,4,5,6)P(5) (Fig. 3B). This identifies the second peak as minimally 88% Ins(1,4,5, 6)P(4). The first InsP(4) peak co-eluted with an internal standard of [^14C]Ins(1,3,4,5)P(4). Upon incubation of the total [^3H]InsP(4) fraction with Ins(1,4,5)P(3)/Ins(1,3,4,5)P(4) 5-phosphatase the first [^3H]InsP(4) peak was totally degraded (Fig. 3C) to a compound with the same retention time as Ins(1,3,4)P(3) (data not shown) identifying the first [^3H]InsP(4) peak as Ins(1,3,4,5)P(4). From these data we concluded that Ins(1,3,4,5,6)P(5) is mainly metabolized to Ins(1,3,4,5)P(4) and Ins(1,4,5,6)P(4). This is a significant conclusion because data summarized in Fig. 1B indicate that both Ins(1,3,4,5)P(4) and Ins(1,4,5,6)P(4) are precursors for material that expressed Ins(1,4,5)P(3) cross-reactivity. It was therefore important to prove that authentic Ins(1,4,5)P(3) was formed from these two InsP(4) isomers.

The metabolism of Ins(1,3,4,5)P(4) was investigated using a mixture of [3-P]Ins(1,3,4,5)P(4) and [^3H]Ins(1,3,4,5)P(4). Incubation of this mixture with particulate fraction resulted in the formation of InsP(3), which had a P:^3H ratio that was 25% that of the original Ins(1,3,4,5)P(4). Since P is removed from the 3-position of Ins(1,3,4,5)P(4), these data demonstrate that 75% of the InsP(3) was Ins(1,4,5)P(3). The remainder of the InsP(3) fraction still contained the P label at the 3-position and was not further identified. In a previous study, using different conditions, the isomers of InsP(3) that were formed were identified as Ins(3,4,5)P(3) and Ins(1,4,5)P(3) (Van Dijken et al., 1995). The metabolism of [^3H]Ins(1,4,5,6)P(4) by particulate fraction was also investigated. The resultant [^3H]InsP(3) fraction was isolated by HPLC, desalted, and incubated with Ins(1,4,5)P(3)/Ins(1,3,4,5)P(4) 5-phosphatase, whereupon 72% of the [^3H]InsP(3) was degraded to InsP(2) (Fig. 4B). Under identical conditions authentic [^3H]Ins(1,4,5)P(3) was completely degraded. This means that 72% of the InsP(3) formed from Ins(1,4,5,6)P(4) is Ins(1,4,5)P(3).


Figure 4: Identification of Ins(1,4,5)P(3) formed from Ins(1,3,4,5)P(4) and Ins(1,4,5,6)P(4) degradation by Dictyostelium particulate fraction. A, [3-P]Ins(1,3,4,5)P(4) (3000 dpm, ) and [^3H]Ins(1,3,4,5)P(4) (7000 dpm, ) were mixed and degraded for 30 min with Dictyostelium particulate fraction (5 times 10^7 lysed cells/ml) as described under ``Experimental Procedures.'' The reaction products were separated on a Zorbax SAX column. The gradient used was 0 min, 0% B; 10 min, 10% B. The P:^3H ratio in the formed InsP(3) fraction is 0.25 relative to this ratio in the Ins(1,3,4,5)P(4) fraction. B, [^3H]Ins(1,4,5,6)P(4) was incubated for 30 min with Dictyostelium particulate fraction (5 times 10^7 lysed cells/ml) as described under ``Experimental Procedures.'' The InsP(3) fraction was isolated, desalted, and incubated for 10 min at 37 °C with (350 dpm, ) or without (box, 300 dpm) 10 µl of purified Ins(1,4,5)P(3)/Ins(1,3,4,5)P(4) 5-phosphatase in a 150-µl incubation containing 50 mM Hepes, pH 7.5, 1 mg/ml BSA, 5 mM MgCl(2), 5 mM 2-mercaptoethanol. The sample was analyzed on a Zorbax SAX column. The gradient used was 0 min, 0% B; 3 min, 20% B; 12 min, 100% B.



Together these data indicate that Ins(1,3,4,5,6)P(5) is degraded to two InsP(4) isomers, Ins(1,3,4,5)P(4) and Ins(1,4,5,6)P(4). These InsP(4) isomers are degraded to predominantly Ins(1,4,5)P(3).

Ins(1,4,5)P(3) Formation by a Purified Hepatic MIPP

The above data indicate that in Dictyostelium Ins(1,3,4,5,6)P(5) is degraded via Ins(1,3,4,5)P(4) and Ins(1,4,5,6)P(4) to Ins(1,4,5)P(3). Two of these reaction steps, namely, Ins(1,3,4,5,6)P(5) 3-phosphatase and Ins(1,3,4,5)P(3) 3-phosphatase, have previously been shown to be performed by a single rat liver enzyme that was recently renamed as multiple inositol polyphosphate phosphatase (MIPP; Craxton et al., 1995). Since MIPP also dephosphorylates Ins(1,4,5,6)P(4) (Nogimori et al., 1991) we have now incubated purified MIPP with [^3H]Ins(1,4,5,6)P(4) and we have purified and identified the [^3H]InsP(3) that was formed as follows. After addition of [^14C]Ins(1,4,5)P(3) the sample was incubated with recombinant Ins(1,4,5)P(3) 3-kinase. Fig. 5shows that the [^3H]InsP(3) was co-phosphorylated with [^14C]Ins(1,4,5)P(3) and that the [^3H]-labeled reaction product co-eluted upon HPLC with the internally generated [^14C]Ins(1,3,4,5)P(4) standard. The inset of Fig. 5further shows that over a range of dilutions of recombinant 3-kinase, the extent of phosphorylation of [^3H]InsP(3) and [^14C]Ins(1,4,5)P(3) were identical. This indicates that the [^3H]InsP(3) formed from [^3H]Ins(1,4,5,6)P(4) was [^3H]Ins(1,4,5)P(3). Fig. 6shows a time course of MIPP-mediated degradation of both Ins(1,3,4,5,6)P(5) and Ins(1,4,5,6)P(4). It shows that Ins(1,3,4,5,6)P(5) is more rapidly degraded than Ins(1,4,5,6)P(4). As the specific activities of both substrates used were identical, this indicates that Ins(1,3,4,5,6)P(5) is a better substrate for MIPP than Ins(1,4,5,6)P(4). The inset of Fig. 6shows that the degradation of Ins(1,3,4,5,6)P(5) and Ins(1,4,5,6)P(4) by MIPP is equally effectively inhibited by increasing concentrations of Ins(1,3,4,5)P(4). This indicates that for MIPP-mediated hydrolysis of Ins(1,3,4,5,6)P(5), Ins(1,4,5,6)P(4), and Ins(1,3,4,5)P(4) a single active site is involved.


Figure 5: Identification of the InsP(3) product of MIPP-catalyzed hydrolysis of Ins(1,4,5,6)P(4). [^3H]Ins(1,4,5,6)P(4) was hydrolyzed by MIPP as described in the legend to Fig. 6, and the [^3H]InsP(3) (1500 dpm, ) was isolated by HPLC, desalted, and incubated after the addition of [^14C]Ins(1,4,5)P(3) (200 dpm, ) for 10 min at 37 °C with a 2500-fold dilution of recombinant rat brain Ins(1,4,5)P(3) 3-kinase exactly as described by Craxton et al.(1994). Reactions were quenched, neutralized, and chromatographed by HPLC as described by Menniti et al.(1990). The 50-110-min portion of the HPLC gradient depicts co-elution of both the unknown [^3H]InsP(3) with the [^14C]Ins(1,4,5)P(3) standard and co-chromatography of the [^3H]InsP(4) product with the internally derived [^14C]Ins(1,3,4,5)P(4) standard. The relative position of a [^3H]Ins(1,3,4,6)P(4) standard in a subsequent HPLC elution profile is indicated by the arrow. Further assays (see inset) contained various dilutions of recombinant Ins(1,4,5)P(3) 3-kinase (100-10,000-fold). Data are expressed as the percentage phosphorylation of either [^14C]Ins(1,4,5)P(3) (closed squares) or [^3H]InsP(3) (open squares). No significant phosphorylation of either [^14C]Ins(1,4,5)P(3) or the unknown [^3H]InsP(3) was observed with control E. coli lysates (100-fold dilution).




Figure 6: Dephosphorylation of Ins(1,4,5,6)P(4) and Ins(1,3,4,5,6)P(5) by purified MIPP. Trace quantities of either [^3H]Ins(1,4,5,6)P(4) (4000 dpm, ) or [^3H]Ins(1,3,4,5,6)P(5) (4000 dpm, bullet) were incubated with purified MIPP (0.95 ng) for the indicated times. The [^3H]Ins(1,3,4,5,6)P(5) used in this experiment was also the precursor of this batch of [^3H]Ins(1,4,5,6)P(4); therefore, the specific activities of these polyphosphates were identical. [^3H]inositol phosphates were separated on Bio-Rad AG1-X8 columns. Data represent averages of duplicate incubations. Inset, inhibition of MIPP-catalyzed dephosphorylation of [^3H]Ins(1,4,5,6)P(4) (4000 dpm, ) and [^3H]Ins(1,3,4,5,6)P(5) (4000 dpm, bullet) by Ins(1,3,4,5)P(4) (0-10 µM). [^3H]inositol phosphates were resolved as described above, and data were expressed as a percentage of MIPP activity in the absence of Ins(1, 3, 4, 5)P(4). The conditions used were such that less than 20% of the substrate was metabolized.



The unexpected demonstration that MIPP could catalyze Ins(1,4,5,6)P(4) 6-phosphatase activity now led us to reconsider the specificity of this enzyme's attack upon Ins(1,3,4,5,6)P(5). Previous experiments only identified an Ins(1,3,4,5,6)P(5) 3-phosphatase activity (Nogimori et al., 1991), but we have now greatly increased the sensitivity of such an assay by incubating MIPP with a large amount (750,000 dpm) of [^3H]Ins(1,3,4,5,6)P(5). Fig. 7shows that in addition to the large amount of [^3H]Ins(1,4,5,6)P(4) that we anticipated would be formed (97% of total [^3H]InsP(4)), small amounts of two additional InsP(4) isomers were detected. One of these (2% of total [^3H]InsP(4)) eluted just before the [^14C]Ins(1,3,4,5)P(4) standard and was therefore identified as [^3H]Ins(1,3,4,6)P(4). Another [^3H]InsP(4) peak (1% of total [^3H]InsP(4)) co-eluted with an internal [^14C]Ins(1,3,4,5)P(4) standard and must therefore be Ins(1,3,4,5)P(4) and/or Ins(1,3,5,6)P(4). These data provide only a minimum estimate of flux through Ins(1,3,4,5)P(4), since this InsP(4) isomer will be further metabolized to Ins(1,4,5)P(3) by MIPP.


Figure 7: InsP(4) isomers formed from MIPP-mediated hydrolysis of [^3H] Ins(1,3,4,5,6)P(5). MIPP (0.4 µg) was incubated for 3 h at 37 °C in 1.33 ml of medium containing 100 mM KCl, 50 mM Bis-Tris, pH 6.1, 1 mM EDTA, 0.5 mM EGTA, 2 mM CHAPS, 0.05% (w/v) BSA, and approximately 750,000 dpm [^3H]Ins(1,3,4,5,6)P(5) (circle). Incubations were quenched with perchloric acid and neutralized with freon/octylamine as described under ``Experimental Procedures.'' Samples were spiked with an internal standard of approximately 400 dpm [^14C]Ins(1,3,4,5)P(4) () and chromatographed by HPLC on an Adsorbosphere SAX column as described by Menniti et al.(1990). The inset shows a magnification of the area indicated with a box.




DISCUSSION

Prior to these studies, the only known de novo source for Ins(1,4,5)P(3)in vivo was phospholipase C-mediated hydrolysis of PtdIns(4,5)P(2). Even though cells contain a variety of highly phosphorylated inositol polyphosphates, there has been general acceptance of the axiom that these compounds ``must be synthesised and degraded without inadvertently generating significant quantities of potent signals such as Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4)'' (Downes and McPhee, 1990). Thus, it was completely unexpected to discover that a Dictyostelium mutant in which the gene for phospholipase C was disrupted and rendered nonfunctional was nevertheless capable of maintaining near normal unstimulated levels of both Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4) (Drayer et al., 1994). This led to the conclusion that there may be a phospholipase C-independent source for these second messengers and therefore initiated an unprecedented search for an alternative pathway by which these compounds could be synthesized. Here we demonstrate that Ins(1,3,4,5,6)P(5) is a physiologically relevant precursor of both Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4) in Dictyostelium. Moreover, we have further demonstrated that the same enzymatic reactions can also occur in rat liver by, somewhat surprisingly, the action of a single enzyme.

The mammalian enzyme that degrades Ins(1,3,4,5,6)P(5) to Ins(1,4,5)P(3) was originally identified as an Ins(1,3,4,5)P(4) 3-phosphatase (Cunha-Melo et al., 1988; Doughney et al., 1988; Höer et al., 1988). Recently, it was suggested that the enzyme be renamed a multiple inositol polyphosphate phosphatase (MIPP) since it also removed the 3-phosphate from Ins(1,3,4,5,6)P(5) and could nonspecifically dephosphorylate InsP(6) (Nogimori et al., 1991; Craxton et al., 1995). Here we show that this enzyme is also capable of hydrolyzing the 6-phosphate of both Ins(1,3,4,5,6)P(5) and Ins(1,4,5,6)P(4). This new observation can be rationalized by noting the similarities of the structures of ``inverted'' Ins(1,4,5,6)P(4) and Ins(1,3,4,5)P(4); the inositol ring pucker is similar in both cases, and the 3-hydroxy, 4-phosphate, 5-phosphate, and 6-phosphate of ``inverted'' Ins(1,4,5,6)P(4), respectively, mimic the configuration of the 6-hydroxy, 5-phosphate, 4-phosphate, and 3-phosphate of Ins(1,3,4,5)P(4) (Fig. 8). Provided these similarities satisfy the requirements for substrate recognition and that the nature and orientation of the C-1 and C-2 substituents are not critical determinants of substrate recognition, the 6-phosphate of Ins(1,4,5,6)P(4) can be considered to mimic the 3-phosphate of Ins(1,3,4,5)P(4). An attack upon the 6-phosphate of Ins(1,3,4,5,6)P(5) can be explained similarly since the 3-phosphate of Ins(1, 3,4,5,6)P(5) is mimicked by the 6-phosphate once the Ins(1,3,4,5,6)P(5) is ``inverted.''


Figure 8: Comparison of the structures of Ins(1,3,4,5)P(4) and ``inverted'' Ins(1,4,5,6)P(4).



The exact relationship between the enzyme activities in Dictyostelium particulate fraction and rat MIPP is still unclear. Despite the striking similarities of the net dephosphorylation of Ins(1,3,4,5,6)P(5) to Ins(1,4,5)P(3) and the similar localization of the enzyme activities to the particulate portion of the cells, major differences between the enzyme activities in Dictyostelium particulate fraction and rat MIPP exist. For example, Ins(1,3,4,5)P(4) is, at best, a relatively minor product to accumulate when MIPP hydrolyzes Ins(1,3,4,5,6)P(5), whereas in Dictyostelium particulate fraction comparable amounts of Ins(1,3,4,5)P(4) and Ins(1,4,5,6)P(4) accumulate. This difference may reflect a similarity in the rates of Dictyostelium 3- and 6-phosphatase activities toward Ins(1,3,4,5,6)P(5), in sharp contrast to the substantial preference of MIPP for the 3-phosphate of Ins(1,3,4,5,6)P(5). The most striking difference lay in the effect of CaCl(2) on the enzyme reaction; the net conversion of Ins(1,3,4,5,6)P(5) to Ins(1,4,5)P(3) by the Dictyostelium phosphatase activities is only detectable in the presence of Ca, but when we investigated the effect of up to 1 mM Ca upon MIPP, the activity was increased by no more than 20% (data not shown).

In order for hepatic MIPP (or any other mammalian species of this enzyme) to produce significant amounts of Ins(1,4,5)P(3) from Ins(1,3,4,5,6)P(5)in vivo, both the enzyme and the Ins(1,3,4,5,6)P(5) would have to be in a compartment that did not have access to even the tiny amounts of InsP(6) that competitively inhibit this enzyme activity (Nogimori et al., 1991). As it happens, in rat liver at least, MIPP resides inside the endoplasmic reticulum (Ali et al., 1993) but we have no evidence that Ins(1,3,4,5,6)P(5) has specific access to the enzyme in vivo. Nevertheless, we should still pursue this possibility, since if it is correct that there is a cellular subcompartment in which a pool of Ins(1,4,5)P(3) can be generated independently of PtdIns(4,5)P(2), this could finally address the longstanding enigma that a number of unstimulated mammalian cell types contain rather more Ins(1,4,5)P(3) than is necessary to fully mobilize Ca stores (see Shears(1992)). We should also consider that there might be physiological relevance to the demonstration that MIPP produced small amounts of Ins(1,3,4,6)P(4) from Ins(1,3,4,5,6)P(5), since just such a reaction was hypothesized to occur in 3T3 fibroblasts (Balla et al., 1994). The evidence for Ins(1,3,4,5,6)P(5) being a physiologically relevant precursor of Ins(1,4,5)P(3) is much stronger in the case of Dictyostelium. For example, there are kinetic differences between the Ins(1,4,5)P(3)-forming activities of MIPP as compared with the Dictyostelium enzyme (see above), and it seems from our data that the latter does not need to be spatially separated from InsP(6) in order to function. More importantly, total steady-state levels of Ins(1,4,5)P(3)in vivo were only slightly decreased by rendering inactive all phospholipase C activity in this organism, indicating that another de novo pathway was present (Drayer et al., 1994). Moreover, previously published data on the inositol phosphates present in intact Dictyostelium cells substantiate the idea that in this organism the conversion of Ins(1,3,4,5,6)P(5) to Ins(1,4,5)P(3) is of physiological relevance. Drayer et al.(1994) showed that, in plc cells that contained near normal levels of Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4), the levels of Ins(1,3,4,5,6)P(5) were specifically reduced by around 70%. Indeed, this reduction in levels of Ins(1,3,4,5,6)P(5) is indicative of an adaptive response in order to sustain the steady-state levels of important signaling molecules such as Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4). Although wild-type Dictyostelium cells contain large quantities of several InsP(5) isomers (Stephens et al., 1991), it is Ins(1,3,4,5,6)P(5) that is formed as an intermediate in the stepwise phosphorylation of inositol to InsP(6) (Stephens and Irvine, 1990). Together these results suggest an independent de novo link between Ins(1,4,5)P(3) and inositol (Fig. 9). This conclusion should also prompt research into the possibility that mammalian tissues might also synthesize some Ins(1,3,4,5,6)P(5) and InsP(6) by this pathway.


Figure 9: The inositol cycle in Dictyostelium discoideum. The metabolism of inositol in the slime mold Dictyostelium is shown. The metabolic steps presented in this study are indicated with broad arrows. For simplicity not all metabolic pathways of PIP and PI are indicated in the figure.



Since the role of Ins(1,4,5)P(3) is itself to mobilize Ca, the observation that the formation of Ins(1,4,5)P(3) from Ins(1,3,4,5,6)P(5) is Ca dependent, raise some hitherto unexpected and complex questions concerning the interactions between levels of Ca and Ins(1,4,5)P(3). It is possible that the calcium-mediated activation of the Dictyostelium enzyme is indicative of an ongoing regulatory process in the cytosol. Certainly the observation that organisms as diverse as Dictyostelium and the rat both possess the enzymatic capacity to synthesize Ins(1,4,5)P(3) from Ins(1,3,4,5,6)P(5), essentially independently of short term PtdIns(4,5)P(2) turnover, should provoke us to reevaluate the cellular control processes that regulate Ca mobilization.


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. This study was supported by the foundation for life sciences (SLW), which is subsidized by the Netherlands Organization for Scientific Research (NWO).

§
To whom correspondence should be addressed: Tel.: 31-50-634173; Fax: 31-50-634165; P.J.M.Van.Haastert@chem.rug.nl.

(^1)
Inositol polyphosphates are abbreviated according to IUPAC nomenclature, where ``Ins'' represents myo-inositol, and the phosphate substituents are as indicated (i.e. Ins(1,4,5)P(3) represents inositol 1,4,5-trisphosphate).

(^2)
The abbreviations used are: PLC, phosphatidylinositol-specific phospholipase C; MIPP, multiple inositol polyphosphate phosphatase; HPLC, high pressure liquid chromatography; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

(^3)
J. Van Der Kaay and P. J. M. Van Haastert, Biochem. J. in press.


ACKNOWLEDGEMENTS

We thank W. D. Freund for providing Paramecium cells.


REFERENCES

  1. Ali, N., Craxton, A., and Shears, S. B. (1993) J. Biol. Chem. 268, 6161-6167 [Abstract/Free Full Text]
  2. Balla, T., Sim, S. S., Baukal, A. J., Rhee, S. G., and Catt, K. J. (1994) Mol. Biol. Cell 5, 17-28 [Abstract]
  3. Bartfai, T. (1979) Adv. Cyclic Nucleotide Res. 10, 219-242 [Medline] [Order article via Infotrieve]
  4. Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205 [CrossRef][Medline] [Order article via Infotrieve]
  5. Bominaar, A. A., and Van Haastert, P. J. M. (1994) Biochem. J. 297, 189-193 [Medline] [Order article via Infotrieve]
  6. Craxton A., Erneux, C., and Shears, S. B. (1994) J. Biol. Chem. 269, 4337-4342 [Abstract/Free Full Text]
  7. Craxton, A., Ali, N., and Shears, S. B. (1995) Biochem. J. 305, 491-498 [Medline] [Order article via Infotrieve]
  8. Cunha-Melo, J. R., Dean, N. M., Ali, H., and Beaven, M. A. (1988) J. Biol. Chem. 263, 14245-14250 [Abstract/Free Full Text]
  9. Das, O. P., and Henderson, E. J. (1983) Biochim. Biophys. Acta 736, 45-56
  10. Doughney, C., McPherson, M. A., and Dormer, R. L. (1988) Biochem. J. 251, 927-929 [Medline] [Order article via Infotrieve]
  11. Downes, C. P., and McPhee, C. H. (1990) Eur. J. Biochem. 195, 1-18
  12. Drayer, A. L., and Van Haastert, P. J. M. (1992) J. Biol. Chem. 267, 18387-18392 [Abstract/Free Full Text]
  13. Drayer, A. L., Van Der Kaay, J., Mayr, G. W., and Haastert, P. J. M (1994) EMBO J. 13, 1601-1609 [Abstract]
  14. Europe-Finner, G. N., and Newell, P. C. (1986) Biochim. Biophys. Acta 887, 335-340 [Medline] [Order article via Infotrieve]
  15. Flaadt, H., Jaworski, E., Schlatterer, C., and Malchow, D. (1993) J. Cell Sci. 105, 255-261 [Abstract/Free Full Text]
  16. Freund, W. D., Mayr, G. W., Tietz, C., and Schultz, J. E. (1992) Eur. J. Biochem. 207, 359-367 [Abstract]
  17. Höer, D., Kwiatkowski, A., Seib, C., Rosenthal, W., Schultz, G., and Oberdisse, E. (1988) Biochem. Biophys. Res. Commun. 154, 668-675 [CrossRef][Medline] [Order article via Infotrieve]
  18. Irvine, R. F. (1992) Adv. Second Messenger Phosphoprotein Res. 26, 161-185 [Medline] [Order article via Infotrieve]
  19. Kirk, C. J., Morris, A. J., and Shears, S. B. (1990) in Peptide Hormone Action : A Practical Approach (Siddle, K., and Hutton. J. C., eds) pp. 151-184, IRL press, Oxford
  20. Menniti, F. S., Oliver, K. G., Nogimori, K., Obie, J. F., Shears, S. B., and Putney, J. W., Jr. (1990) J. Biol. Chem. 265, 11167-11176 [Abstract/Free Full Text]
  21. Nogimori, K., Hughes, P. J., Glennon, M. C., Hodgson, M. E., Putney, J. W., Jr., and Shears, S. B. (1991) J. Biol. Chem. 266, 16499-16506 [Abstract/Free Full Text]
  22. Shears, S. B. (1992) Adv. Second Messenger Phosphoprotein Res. 26, 63-92 [Medline] [Order article via Infotrieve]
  23. Stephens, L. R., and Downes, C. P. (1990) Biochem. J. 265, 435-452 [Medline] [Order article via Infotrieve]
  24. Stephens, L. R., and Irvine, R. F. (1990) Nature 346, 580-582 [CrossRef][Medline] [Order article via Infotrieve]
  25. Stephens, L. R., Hawkins, P. T., Stanley, A. F., Moore, T., Poyner, D. R., Morris, P. J., Hanley, M. R., Kay, R. R., and Irvine, R. F. (1991) Biochem. J. 275, 485-499 [Medline] [Order article via Infotrieve]
  26. Takazawa, K., and Erneux, C. (1991) Biochem. J. 280, 125-129 [Medline] [Order article via Infotrieve]
  27. Takazawa, K., Vanderkerckhove, J., Dumont, J. E., and Erneux, C. (1990) Biochem. J. 272, 107-112 [Medline] [Order article via Infotrieve]
  28. Van Der Kaay, J., and Van Haastert, P. J. M. (1995) Anal. Biochem. 225, 183-185 [CrossRef][Medline] [Order article via Infotrieve]
  29. Van Dijken, P., Lammers, A. A., Ozaki, S., Potter, B. V. L., Erneux, C., and Van Haastert, P. J. M. (1994) Eur. J. Biochem. 226, 561-566 [Abstract]
  30. Van Dijken, P., Lammers, A. A., and Van Haastert, P. J. M (1995) Biochem. J. 308, 127-130 [Medline] [Order article via Infotrieve]
  31. Van Haastert, P. J. M. (1989) Anal. Biochem. 177, 115-119 [Medline] [Order article via Infotrieve]
  32. Van Lookeren Campagne, M. M., Erneux, C., Van Eijk, R., and Van Haastert, P. J. M. (1988) Biochem. J. 254, 343-350 [Medline] [Order article via Infotrieve]
  33. Verjans, B., Moreau, C., and Erneux, C. (1994) Mol. Cell. Endocrinol. 98, 167-171 [CrossRef][Medline] [Order article via Infotrieve]
  34. Ye, W., Ali, N., Bembenek, M. E., Shears, S. B., and Lafer, E. M. (1995) J. Biol. Chem. 270, 1564-1568 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.