JBC Invitrogen Ultrasensitive Cytokine Assays

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J Biol Chem, Vol. 274, Issue 27, 18973-18980, July 2, 1999


Inositol 1,3,4-Trisphosphate Acts in Vivo as a Specific Regulator of Cellular Signaling by Inositol 3,4,5,6-Tetrakisphosphate*

Xiaonian YangDagger §, Marco Rudolf, Mark A. CarewDagger , Masako YoshidaDagger , Volkmar Nerreter, Andrew M. Rileyparallel , Sung-Kee Chung**, Karol S. BruzikDagger Dagger , Barry V. L. Potterparallel , Carsten Schultz, and Stephen B. ShearsDagger

From the Dagger  Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, the  Institut für Organische Chemie, Universität Bremen, 28359 Bremen, Germany, the parallel  Wolfson Laboratory for Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom, the Dagger Dagger  Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Chicago, Illinois 60612-7231, ** Department of Chemistry, Pohang University of Science and Technology, San 31 Hyoja Dong, Pohang 790-784, Korea

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ca2+-activated Cl- channels are inhibited by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4) (Xie, W., Kaetzel, M. A., Bruzik, K. S., Dedman, J. R., Shears, S. B., and Nelson, D. J. (1996) J. Biol. Chem. 271, 14092-14097), a novel second messenger that is formed after stimulus-dependent activation of phospholipase C (PLC). In this study, we show that inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) is the specific signal that ties increased cellular levels of Ins(3,4,5,6)P4 to changes in PLC activity. We first demonstrated that Ins(1,3,4)P3 inhibited Ins(3,4,5,6)P4 1-kinase activity that was either (i) in lysates of AR4-2J pancreatoma cells or (ii) purified 22,500-fold (yield = 13%) from bovine aorta. Next, we incubated [3H]inositol-labeled AR4-2J cells with cell permeant and non-radiolabeled 2,5,6-tri-O-butyryl-myo-inositol 1,3,4-trisphosphate-hexakis(acetoxymethyl) ester. This treatment increased cellular levels of Ins(1,3,4)P3 2.7-fold, while [3H]Ins(3,4,5,6)P4 levels increased 2-fold; there were no changes to levels of other 3H-labeled inositol phosphates. This experiment provides the first direct evidence that levels of Ins(3,4,5,6)P4 are regulated by Ins(1,3,4)P3 in vivo, independently of Ins(1,3,4)P3 being metabolized to Ins(3,4,5,6)P4. In addition, we found that the Ins(1,3,4)P3 metabolites, namely Ins(1,3)P2 and Ins(3,4)P2, were >100-fold weaker inhibitors of the 1-kinase compared with Ins(1,3,4)P3 itself (IC50 = 0.17 µM). This result shows that dephosphorylation of Ins(1,3,4)P3 in vivo is an efficient mechanism to "switch-off" the cellular regulation of Ins(3,4,5,6)P4 levels that comes from Ins(1,3,4)P3-mediated inhibition of the 1-kinase. We also found that Ins(1,3,6)P3 and Ins(1,4,6)P3 were poor inhibitors of the 1-kinase (IC50 = 17 and >30 µM, respectively). The non-physiological trisphosphates, D/L-Ins(1,2,4)P3, inhibited 1-kinase relatively potently (IC50 = 0.7 µM), thereby suggesting a new strategy for the rational design of therapeutically useful kinase inhibitors. Overall, our data provide new information to support the idea that Ins(1,3,4)P3 acts in an important signaling cascade.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

There is considerable interest in the idea that Ins(1,4,5)P3 1 and Ins(1,3,4,5)P4 (Fig. 1) act in a co-ordinated manner as mediators of stimulus-dependent Ca2+ mobilization (1, 2). This has naturally led us to consider that the 5-phosphatases that degrade Ins(1,4,5)P3 and Ins(1,3,4,5)P4 (3) are signaling "off-switches." This in turn has created the impression that the pathway by which these two inositol phosphates are dephosphorylated serves only as a conduit that replenishes the free inositol pool. In contrast, we have recently suggested that one of these downstream products, namely Ins(1,3,4)P3, should be viewed in an important cell-signaling context (4). This new hypothesis comes from the observation that a rat hepatic Ins(3,4,5,6)P4 1-kinase was inhibited in vitro by Ins(1,3,4)P3 (4, 5). The reason that this effect of Ins(1,3,4)P3 upon Ins(3,4,5,6)P4 metabolism is of such interest is that Ins(3,4,5,6)P4 is an inhibitor of the conductance of the calcium-activated Cl- channels in the plasma membrane (6-9). These ion channels make important contributions to salt and fluid secretion, and in addition they may participate in osmoregulation, pH balance, and smooth muscle excitability (10-13).

The cellular accumulation of Ins(3,4,5,6)P4 is known to correlate well with receptor-dependent changes in PLC activity, but the molecular mechanisms that link these two events have not been fully elucidated (14). Our current hypothesis (15, 16) is that cellular levels of Ins(3,4,5,6)P4 depend upon a dynamic balance between two competing enzyme activities acting in a closed substrate cycle: Ins(1,3,4,5,6)P5 1-phosphatase and Ins(3,4,5,6)P4 1-kinase (Fig. 1). The poise of this cycle is proposed to be regulated in such a manner that it can shift in favor of Ins(3,4,5,6)P4 accumulation whenever PLC is activated, perhaps through inhibition of the Ins(3,4,5,6)P4 1-kinase by Ins(1,3,4)P3 (Fig. 1). However, to date such inhibition has only been observed in studies with the purified rat hepatic kinase (4, 5). No direct evidence has previously been published that indicates Ins(1,3,4)P3 can regulate Ins(3,4,5,6)P4 1-kinase activity in intact cells; it was a goal of the current study to explore this issue.

In order to investigate if Ins(1,3,4)P3 can regulate Ins(3,4,5,6)P4 levels in intact cells, it was necessary to increase cellular levels of Ins(1,3,4)P3 specifically, under conditions where PLC activity was not activated. In this way, we could avoid the possibility of Ins(3,4,5,6)P4 metabolism also being regulated by the many additional signal transduction processes that are activated downstream of PLC. To this end, the development of cell-permeant, bioactivatable analogues of inositol phosphates (7, 17, 18) has provided us with new opportunities to examine the functions of inositol polyphosphates in intact cells. The charge-masking groups that enable these derivatives to permeate into cells are hydrolyzed by intracellular esterases, releasing the native isomer (7, 17). In this study we used a new cell-permeant analogue, 2,5,6-tri-O-butyryl-myo-inositol 1,3,4-trisphosphate hexakis(acetoxymethyl) ester (Bt3Ins(1,3,4)P3/AM; Ref. 19), to elevate the cellular concentration of Ins(1,3,4)P3 inside rat pancreatoma (AR4-2J) cells.

This experimental approach was important for another reason. Rather than Ins(1,3,4)P3 regulating an enzyme of Ins(3,4,5,6)P4 metabolism (Fig. 1), in principle, Ins(1,3,4)P3 could instead elevate Ins(3,4,5,6)P4 concentration simply by being metabolized to it (i.e. a mass action effect). For example, Ins(1,3,4)P3 can be converted to Ins(3,4,5,6)P4 by the sequential actions of Ins(1,3,4)P3 6-kinase, Ins(1,3,4,6)P4 5-kinase, and Ins(1,3,4,5,6)P5 1-phosphatase (15, 16, 20). Others have proposed an alternative pathway for de novo Ins(3,4,5,6)P4 synthesis, which requires the sequential actions of Ins(1,3,4)P3 6-kinase, Ins(1,3,4,6)P4 1-phosphatase, and Ins(3,4,6)P3 5-kinase (21-23). We have now used AR4-2J cells to examine whether Ins(1,3,4)P3 alters Ins(3,4,5,6)P4 levels by mass action effects. Our strategy was based upon first prelabeling the metabolic pool of Ins(3,4,5,6)P4 to steady-state with [3H]inositol. These cells were then treated with non-radiolabeled Bt3Ins(1,3,4)P3/AM. We investigated if there was any significant metabolic flux from Ins(1,3,4)P3 to Ins(3,4,5,6)P4, which would have revealed itself by tending to decrease the amount of 3H label in the [3H]Ins(3,4,5,6)P4 pool, due to a pulse-chase effect (15).


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  		Fig. 1.   Proposed mechanism for the regulation of cellular Ins(3,4,5,6)P4 levels by Ins(1,3,4)P3. The proposed link between Ins(1,3,4)P3 levels and the interconversion of Ins(1,3,4,5,6)P5 and Ins(3,4,5,6)P4 (15, 16) is described. The broken line illustrates the inhibitory effect of Ins(1,3,4)P3 upon Ins(3,4,5,6)P4 phosphorylation by the 1-kinase.

Another feature of an effective signal transduction process relates to its specificity. If the biological effects of a signaling compound cannot be imitated by its products and precursors, this provides sensitivity in the signaling "on" and "off" switches. In the case of signaling by Ins(1,3,4)P3, the "on-switch" is dephosphorylation of Ins(1,3,4,5)P4 (3). This process is particularly sensitive, as Ins(1,3,4,5)P4 is a 290-fold weaker inhibitor of the 1-kinase than is Ins(1,3,4)P3 (4). We have now turned our attention to considering how effective is the dephosphorylation of Ins(1,3,4)P3 as a signaling off-switch. In vivo, both 4- and 1-phosphatases actively degrade Ins(1,3,4)P3 to Ins(1,3)P2 and Ins(3,4)P2, respectively (24-26). We have therefore determined the potency with which these bisphosphate degradation products inhibit the 1-kinase.

There was one further aspect to this study that is relevant to the development of Ins(3,4,5,6)P4 agonists and antagonists for pharmacological intervention in the signaling actions of Ins(3,4,5,6)P4 (18, 27). This goal is directed at diseases that might be treated by either up-regulating or down-regulating Ca2+-activated Cl- secretion (18, 27). A major challenge to pharmacological intervention at the effector site for Ins(3,4,5,6)P4 comes from the exquisite specificity with which it blocks Cl- channel conductance; Ins(1,3,4)P3, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, and Ins(1,3,4,5,6)P5 are all ineffective (6, 8, 9). Moreover, at least one, and possibly both of the OH groups on Ins(3,4,5,6)P4, are also critical determinants of substrate specificity (18, 28). This tight specificity may make it difficult to rationally design a functional analogue of Ins(3,4,5,6)P4. A possible alternative therapeutic strategy might be to target Ins(3,4,5,6)P4 synthesis, rather than its site of action. To this end, we examined the impact on the 1-kinase of some analogues of Ins(1,3,4)P3 that contain a phosphate group in the 2-position.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- [3H]Inositol was purchased from American Radiolabeled Chemicals Inc. or NEN Life Science Products. [3H]Ins(1,3,4)P3 was prepared by dephosphorylating [3H]Ins(1,3,4,5)P4 (20 Ci/mmol, NEN Life Science Products) with recombinant Ins(1,3,4,5)P4 5-phosphatase, which was kindly provided by Dr. C. Erneux (29). Ins(1,4)P2 was purchased from Sigma. D/L-2,5,6-Tri-O-butyryl-myo-inositol 1,3,4-trisphosphate-hexakis(acetoxymethyl) ester (D/L-Bt3Ins(1,3,4)P3/AM) was synthesized as described previously (19). In some experiments, we used enantiomerically pure D-Bt3Ins(1,3,4)P3/AM. This compound was prepared from the enantiomerically pure precursor, 4-O-benzyl-1,2:5,6-di-O-cyclohexylidene-myo-inositol (30). Alkylation of the hydroxy group with benzyl bromide in dimethyl formamide at 50 °C for 20 h and in the presence of an excess of sodium hydride and tetrabutyl ammonium iodide afforded 3,4-di-O-benzyl-1,2:5,6-di-O-cyclohexylidene-myo-inositol. Purification by preparative HPLC (92% MeOH, RP-18, 10 µm, 50 × 250 mm, 40 ml/min) gave 55% yield as an oil [alpha ]D20 was +23°; c = 0.75, chloroform). The more labile ketal was removed by a 2-h treatment with acetyl chloride (5%) in a mixture of acetonitrile and methanol (4:5, v/v). The solution was neutralized with triethylamine and evaporated to dryness. The crude material was purified by preparative HPLC (90% MeOH, RP-18, 10 µm, 50 × 250 mm, 40 ml/min) to give 3,4-di-O-benzyl-1,2-O-cyclohexylidene-myo-inositol in 58% yield as a clear oil [alpha ]D20 was -11.1°; c = 0.72, chloroform). The latter was finally converted to D-Bt3Ins(1,3,4)P3/AM, as described previously for the racemic precursor (19). Analytical data for D-Bt3Ins(1,3,4)P3/AM were as follows: [alpha ]D20 was -7.3° (c = 0.87, toluene). Direct chemical ionization high resolution mass spectroscopy [M-CH2OAc]- (C33H52O28P3) gave a calculated m/z of 989.1858; found m/z was 989.1868. 1H and 31P NMR data were in accordance with those of the racemic compound. Enantiomerically pure D-Bt2Ins(1,4,5,6)P4/AM was synthesized as described previously (7). All bioactivatable esters were dissolved in Me2SO/Pluronic (5%, v/v) as described previously (7).

Ins(1,3)P2, D/L-Ins(3,4)P2, D/L-Ins(1,2,4)P3, Ins(1,2,3)P3, D/L-Ins(1,2,4,6)P4, and D/L-Ins(1,2,3,4)P4 were prepared as described previously (31-33). Ins(1,3,6)P3 and Ins(1,4,6)P3 were prepared as described previously (34, 35). Sources of other inositol phosphates are given elsewhere (4, 8).

Bombesin, bovine serum albumin, phosphocreatine, phosphocreatine kinase, heparin agarose resin (type II and IIIs), and protease inhibitors were purchased from Sigma. The calmodulin-dependent protein kinase (CaM KII) was obtained from New England Biolabs. Protein kinases A and C, and their assay kits (SpinZyme Format), were the products of Pierce. The UNO Q12 anion exchange column was acquired from Bio-Rad Laboratories. Polyethylene glycol 4000 was purchased from Fluka. Frozen bovine aorta were purchased from Pel-Freez Biological.

Assay of Ins(3,4,5,6)P4 1-Kinase-- The 1-kinase activity was assayed as described before (4). Briefly, 10-20 µl of enzyme was incubated at 37 °C in a final volume of 100 µl containing about 4000 dpm [3H]Ins(3,4,5,6)P4, which was adjusted to a concentration of 0.25 µM with non-radioactive substrate, 20 mM HEPES (pH 7.2), 6 mM MgSO4, 0.4 mg/ml saponin, 100 mM KCl, 0.3 mg/ml bovine serum albumin, 2 µM InsP6, 5 mM ATP, 10 mM phosphocreatine, and 2.5 Sigma units of phosphocreatine kinase. After 30 min., the reaction was stopped by quenching with 1 ml of ice-cold medium containing 1 mg/ml InsP6, 0.2 M ammonium formate, and 0.1 M formic acid. The quenched reactions were diluted to 10 ml with deionized water, and chromatographed on Bio-Rad gravity-fed columns using AG 1-X8 ion exchange resin.

For some assays, the 1-kinase was preincubated at 30 °C for 10 min with (a) 125 units of the catalytic subunit of protein kinase A, (b) 0.2 unit of protein kinase C, (c) 600 units of calmodulin, or (d) 500 units of CaM KII, preactivated with calmodulin/Ca2+ (New England Biolabs). The protein kinases used in these experiments were all shown to be active in control experiments (assay kits for protein kinases A and C were supplied by Pierce; the CaM KII was checked using a kit purchased from Upstate Biochemicals).

The 1-kinase was also used as a diagnostic tool to verify the nature of HPLC-purified [3H]Ins(3,4,5,6)P4. In these incubations, 45 µl of purified 1-kinase was added to 225 µl of medium containing 67 mM HEPES (pH 8.0 with KOH), 0.7 mM EDTA, 8.7 mM MgSO4, 6.7 mM ATP, 13.3 mM phosphocreatine, 1.33 µM InsP6, and 6 Sigma units of phosphocreatine kinase. Then, 30 µl of the appropriate HPLC fraction was added (which brought the final pH to approximately 6.5). Reactions (at 37 °C) were allowed to proceed to completion (over a 3-h period), and then the amount of [3H]InsP5 formed was determined using gravity-fed ion-exchange columns, as described above.

Purification of Ins(3,4,5,6)P4 1-Kinase-- Frozen bovine aortas were thawed on ice, the attached fat was removed, and then the aorta were pulverized in a meat grinder. In a typical preparation, 300-350 g of ground aortas were homogenized in two volumes of 50 mM bis-Tris (pH 7.0), 1 mM EGTA, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride in a tissue blender.

The homogenate was filtered through four layers of cheesecloth, and a 10-30% (w/v) polyethylene glycol 4000 precipitate was prepared. The resultant pellet was resuspended in 100 ml of Buffer A containing 50 mM bis-Tris (pH 7.0), 1 mM EGTA, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A. The suspension was filtered and loaded at a flow rate of 1 ml/min onto a heparin-agarose type II column (3.2 × 24 cm). After washing with 300 ml of Buffer A at flow rate of 1.5 ml/min, the bound protein was eluted with a linear gradient of 0-30 mM of sodium pyrophosphate in Buffer A.

The peak fractions of enzyme activity eluted from the heparin column were pooled, then frozen and stored at -70 °C. Either two or three preparations were subsequently thawed and combined, dialyzed against 2 liters of 25 mM bis-Tris (pH 7.0) at 4 °C for 3 h, and loaded onto a UNO Q12 anion exchange column (1.5 × 6.8 cm), which was pre-equilibrated with 100 ml of Buffer A. A constant flow rate of 0.5 ml/min was maintained throughout the chromatography. After washing with 60 ml of Buffer A, the bound protein was eluted with a linear gradient of Buffer A plus 0-300 mM NaCl, followed by 60 ml of Buffer A plus1 M NaCl.

Peak fractions of enzyme activity eluted from the UNO Q12 column were pooled, dialyzed against 2 liters of 25 mM bis-Tris (pH 7.0) at 4 °C for 3 h, and loaded on to heparin-agarose type IIIs (1.1 × 13.5 cm), which was pre-equilibrated with 50 ml of Buffer A. A constant flow rate of 0.5 ml/min was maintained throughout. After washing with 60 ml of Buffer A, the bound protein was eluted with a linear gradient of 0-300 mM NaCl in Buffer A, followed by 60 ml of 1 M NaCl in Buffer A.

The protein concentration of the 1-kinase preparation was determined using Bio-Rad Protein Assay Dye Reagent with bovine serum albumin as standard. Final enzyme preparations were stored in 10% glycerol plus 1 mg/ml bovine serum albumin at -70 °C.

Gel Filtration-- A 1-ml aliquot of a resuspension of a 10-30% PEG precipitation was loaded at a flow rate of 0.25 ml/min to Sephacryl S100 column (2.0 × 86 cm), which was pre-equilibrated with 600 ml of bis-Tris buffer containing 50 mM bis-Tris (pH 7.0), 1 mM EGTA, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 100 mM NaCl. The protein was then chromatographed using the same buffer at a constant 0.25 ml/min flow rate. Fractions (5ml) were collected and assayed for enzyme activity. The column was calibrated under the exactly same conditions using bovine serum albumin, chicken ovalbumin, equine myoglobin, and vitamin B-12.

Culturing and Incubation of AR4-2J Cells-- The AR4-2J pancreatoma cells were cultured in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 10% fetal bovine serum, 2 mM glutamine, 500 units/ml penicillin, and 500 µg/ml streptomycin, with 10% conditioned medium, and harvested by brief trypsinization. Either 2.0 × 105 or 1.2 × 106 cells were seeded in 24-well or 6-well tissue culture plates, respectively. Cells were labeled with 75-150 µCi/ml [3H]myo-inositol for 4 days (medium was replaced on the 3rd day) in 700 µl (for 24-well plates) or 3 ml (for 6-well plates) of the above culture medium. After completion of the labeling protocol, the culture medium was aspirated and the cells were washed twice with Krebs/Ringer/HEPES solution (15). Cells were then incubated in 300 µl (for 24-well) or 1 ml (for 6-well) of Krebs/Ringer/HEPES solution for 2 h. Then 20 mM LiCl was added, and 20 min later cells were treated for the indicated time with (i) a cell-permeant inositol phosphate, (ii) vehicle, or (iii) bombesin.

Cells were quenched and neutralized, and the inositol phosphates were separated by HPLC as described elsewhere (36). Radioactivity was either counted on-line, using a Radiomatic Flo-1, or recovered in 1-ml fractions. The levels of 3H-labeled inositol phosphates were normalized as a ratio to cellular levels of [3H]InsP6; the latter were unaffected by any of the experimental protocols performed in this study.

For some experiments, after the AR4-2J cells were harvested, cells were collected by centrifugation in serum-containing culture medium. The pellet was washed in HEPES-buffered saline, and then a lysate was prepared by resuspending the packed cells in an equal volume of ice-cold lysis buffer comprising: 50 mM KCl, 50 mM HEPES (pH 7.2), 1 mM EDTA, 5 mM ATP, 4 mM CHAPS, 0.4 mM phenylmethylsulfonyl fluoride, 40 µM E-64, 10 µM leupeptin, 3 µM pepstatin.

Assay of Mass Levels of Ins(1,3,4)P3 inside AR4-2J Cells-- Following the HPLC fractionation of extracts of [3H]inositol-labeled cells (see above), 1-ml fractions were saved, from which 25-µl aliquots were counted for radioactivity so as to identify the Ins(1,3,4)P3 peak (because of the low levels of endogenous [3H]Ins(1,3,4)P3, samples were "spiked" with 4000 dpm [3H]Ins(1,3,4)P3 (20 Ci/mmol) before they were applied to the HPLC column). The Ins(1,3,4)P3 peak was then desalted (37) and resuspended in 60 µl Ins(1,3,4)P3 6-kinase assay buffer: 50 mM KCl, 50 mM HEPES, pH 7.2, 10 mM phosphocreatine, 6 mM ATP, 8 mM MgSO4, 25 Sigma units/ml phosphocreatine kinase, 0.5 mg/ml bovine serum albumin. Recovery of [3H]Ins(1,3,4)P3 from the cell extract was typically 70-75%. Each Ins(1,3,4)P3 sample was then divided into two equal portions, named A and B. The Ins(1,3,4)P3 was depleted from portion B by its incubation for 60 min at 37 °C with 0.1 µg of recombinant Ins(1,3,4)P3 1-phosphatase (kindly supplied by Dr. J. York, Duke University, Durham, NC). Control experiments showed that the extent of Ins(1,3,4)P3 hydrolysis exceeded 95%. The Ins(1,3,4)P3 1-phosphatase was then heat-inactivated (3 min at 100 °C). Aliquots (9 µl) of either portion A or B were then incubated in triplicate for 20 min in a total volume of 50 µl containing purified Ins(1,3,4)P3 6-kinase, its assay buffer (see above), plus approximately 4000 dpm [3H]Ins(1,3,4)P3. Other incubations (in quadruplicate) were performed that, in place of the Ins(1,3,4)P3 from a cell extract, contained between 0.1 and 50 pmol of non-radiolabeled Ins(1,3,4)P3. A standard curve was constructed from the decrease in the phosphorylation of [3H]Ins(1,3,4)P3 to [3H]Ins(1,3,4,6)P4 that was observed as the Ins(1,3,4)P3 mass was increased. The difference in Ins(1,3,4)P3 mass values between portions A and B (i.e. the amounts hydrolyzed by the Ins(1,3,4)P3 1-phosphatase) were taken to represent the quantity of Ins(1,3,4)P3 in the original HPLC-purified sample.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Purification and Properties of the Ins(3,4,5,6)P4 1-Kinase from Bovine Aorta-- In our earlier study with the Ins(3,4,5,6)P4 1-kinase in rat liver, the enzyme was found to be extremely labile, and we were only able to elicit a 1600-fold purification with a 1% yield (4). No other laboratory has published a purification protocol for this enzyme. We developed a new strategy for the current study, the most notable aspect of which was the efficiency of an affinity purification step using heparin IIIS (Fig. 2; Table I). Thus, using homogenates of bovine aortas as starting material, we purified the 1-kinase 22,500-fold with a 13% yield (Fig. 2; Table I). Our preparations of 1-kinase have an affinity for Ins(3,4,5,6)P4 (0.1-0.2 µM, data not shown) that is very similar to the substrate affinity of the rat hepatic enzyme (4). Gel filtration indicated the size of the enzyme to be 46 kDa (Fig. 3), which is also similar to that of the rat liver enzyme (4). The 1-kinase was strongly inhibited by Ins(1,3,4)P3 (IC50 = 0.17 µM, Fig. 4A). The enantiomer of Ins(1,3,4)P3, namely Ins(1,3,6)P3, was a 100-fold weaker inhibitor of the Ins(3,4,5,6)P4 1-kinase (Table II). The activity of the purified Ins(3,4,5,6)P4 1-kinase was unaffected when 20 mM KCl in the incubation buffer was substituted with 20 mM LiCl (data not shown).


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  		Fig. 2.   Purification of Ins(3,4,5,6)P4 1-kinase. The 1-kinase was purified as described under "Experimental Procedures" by subjecting a 10-30% PEG precipitate to Heparin II-agarose affinity chromatography (panel A), followed by UNO Q12 anion exchange chromatography (panel B) and Heparin IIIS affinity chromatography (panel C). Data are representative of three experiments.

                              
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Table I
Purification of Ins(3,4,5,6)P4 1-kinase
See under "Experimental Procedures" for details of the purification procedures.


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  		Fig. 3.   Molecular size of the Ins(3,4,5,6)P4 1-kinase. The 1-kinase was analyzed by size-exclusion chromatography as described under "Experimental Procedures". Enzyme activity is indicated by the circles; the squares depict the elution positions of standards: serum albumin albumin (66 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa), and vitamin B-12 (1.35 kDa).


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  		Fig. 4.   Inhibition, by Ins(1,3,4)P3, of the Ins(3,4,5,6)P4 1-kinase from bovine aorta and AR4-2J pancreatoma cells. Panel A, Ins(3,4,5,6)P4 1-kinase activity was assayed as described under "Experimental Procedures," using either the enzyme purified from bovine aorta incubated with the indicated concentrations of Ins(1,3,4)P3 (n = 3, vertical bars denote standard errors) (panel A), or lysates of AR4-2J cells in medium containing either 10 µM Ins(1,3,4)P3 (open circles) or no Ins(1,3,4)P3 (closed circles) (panel B); a single experiment is shown, representative of two.

                              
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Table II
Relative potencies of inositol phosphates as inhibitors of the Ins(3,4,5,6)P4 1-kinase
The potencies with which various inositol phosphates inhibited the Ins(3,4,5,6)P4 1-kinase were determined as described under "Experimental Procedures." Data are presented as IC50 values (means and standard errors, with numbers of experiments in parentheses).

The purified 1-kinase was reconstituted with either protein kinase A, protein kinase C, Ca2+/calmodulin, or CaM KII. In no case was there any modification to 1-kinase activity, nor was there any effect upon the potency of inhibition by Ins(1,3,4)P3 (data not shown). Positive controls for each of these protein kinases were obtained by verifying their activities using appropriate assay kits (see "Experimental Procedures").

The Effect of Bt3Ins(1,3,4)P3/AM upon [3H]Inositol Phosphates in AR4-2J Cells-- One aim of this study was to use intact cells to examine the physiological relevance of the inhibition of the Ins(3,4,5,6)P4 1-kinase by Ins(1,3,4)P3, since this effect has only previously been observed with the isolated enzyme (4, 5). For these experiments we used a cell-permeant and bioactivatable analogue of Ins(1,3,4)P3. There are several important aspects of the experimental protocol that should be emphasized. First, we chose to perform these studies with AR4-2J pancreatoma cells. This decision reflects the fact that, in these cells, Ins(1,4,5)P3 is primarily metabolized by the 5-phosphatase pathway; the Ins(1,4,5)P3 3-kinase is a quantitatively minor metabolic route (38, 39), so the ensuing downstream products, including Ins(1,3,4)P3, are present at unusually low levels in "resting" cells (15). This low base line improves the "signal-to-noise" ratio, making our experiments more sensitive to small experimental manipulations of Ins(1,3,4)P3 levels. We also checked that the Ins(3,4,5,6)P4 1-kinase in these cells was sensitive to inhibition by Ins(1,3,4)P3. In cell lysates, 10 µM Ins(1,3,4)P3 inhibited the rate of phosphorylation of Ins(3,4,5,6)P4 by greater than 95% (Fig. 4B).

A second aspect of our experimental protocol that is worth emphasizing is that we preincubated cells with [3H]inositol for 4 days. At this point the cellular pool of Ins(3,4,5,6)P4 was radiolabeled to steady state (16, 39). Thus, any increases in [3H]Ins(3,4,5,6)P4 that we observed truly reflect elevated mass levels of this polyphosphate. Third, we were concerned that cell-permeant analogues of inositol phosphates are typically de-esterified relatively slowly (17), such that a rapid rate of Ins(1,3,4)P3 dephosphorylation would act to prevent the accumulation of this compound inside cells. Our cell incubation medium was therefore supplemented with lithium, so as to inhibit the Ins(1,3,4)P3 1-phosphatase (40). However, it should be noted that this is only a partial solution of this particular problem, since lithium does not inhibit the less active, alternative pathway of Ins(1,3,4)P3 dephosphorylation by a 4-phosphatase (41). Control experiments indicated that this lithium treatment did not affect levels of [3H]Ins(3,4,5,6)P4 (data not shown).

Extracts of [3H]inositol-labeled control cells were resolved by HPLC, and the various 3H-labeled inositol phosphates were assayed using an on-line scintillation counter (Fig. 5, upper panel). We also analyzed extracts from cells treated with 200 µM D/L-Bt3Ins(1,3,4)P3/AM (Fig. 5, lower panel). In these experiments, any metabolic conversion of Ins(1,3,4)P3 to Ins(3,4,5,6)P4 would, by a pulse-chase effect, tend to decrease the amount of [3H]label in the Ins(3,4,5,6)P4 pool (15). In fact, the opposite result was obtained; the size of the [3H]Ins(3,4,5,6)P4 peak increased about 2-fold (Fig. 5, Table III). The elevation in [3H]Ins(3,4,5,6)P4 levels in our experiments (Fig. 5) cannot be caused by an increased flux of non-radiolabeled Ins(1,3,4)P3 into the [3H]Ins(3,4,5,6)P4 metabolic pool. Note also that there were no significant changes in the sizes of other [3H]inositol-labeled peaks after treatment with D/L-Bt3Ins(1,3,4)P3/AM. To control for the possibility of nonspecific effects of cell-permeant inositol phosphates, we also incubated AR4-2J cells with 200 µM D-Bt2Ins(1,4,5,6)P4/AM (which does not affect 1-kinase activity, see below), and this had no effect upon the [3H]inositol-polyphosphate profiles (data not shown).


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  		Fig. 5.   The effect of D/L-Bt3 Ins(1,3,4)P3/AM upon 3H-labeled inositol phosphates in AR4-2J cells. [3H]Inositol-labeled AR4-2J cells were incubated with D/L-Bt3Ins(1,3,4)P3/AM or vehicle for 60 min, and then the cells were quenched and the inositol phosphates resolved by HPLC, as described under "Experimental Procedures." The data are from one of four representative experiments. The identities of the various peaks (see Ref. 36) are as follows: A, GroPIns; B, Ins1P; C, Ins4P; D, InsP2; E, Ins(1,4,5)P3; F, Ins(1,3,4,6)P4; G, Ins(3,4,5,6)P4; H, InsP5; I, InsP6. The integrated value of the Ins(3,4,5,6)P4 peaks were 640 dpm (upper panel, vehicle) and 1505 dpm (lower panel, D/L-Bt3Ins(1,3,4)P3/AM-treated).

                              
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Table III
The effect of D/L-Bt3Ins(1,3,4)P3/AM upon levels of 3H-labeled inositol phosphates in AR4-2J cells
Data are collated from four experiments with control and D/L-Bt3Ins(1,3,4)P3/AM-treated cells, performed as described by the legend to Fig. 5. Data are expressed as a ratio to [3H]InsP6 (see "Experimental Procedures"). * indicates the effect of D/L-Bt3Ins(1,3,4)P3/AM upon [3H]Ins(3,4,5,6)P4 levels was statistically significant (p = 0.003).

We were only able to synthesize limited amounts of Bt3Ins(1,3,4)P3/AM, and so we did not have sufficient material to perform detailed dose-response curves or time courses. However, we did observe that the treatment of cells with 400 µM D/L-Bt3Ins(1,3,4)P3/AM approximately doubled the elevation in [3H]Ins(3,4,5,6)P4 levels, compared with the effect of 200 µM D/L-Bt3Ins(1,3,4)P3/AM; 20 µM D/L-Bt3Ins(1,3,4)P3/AM did not affect [3H]Ins(3,4,5,6)P4 levels (data not shown). One factor that must be taken into account when using these types of cell-permeant analogues is the relatively slow rate of their activation by intracellular esterases (17).

The Ins(3,4,5,6)P4 1-kinase was used as a diagnostic tool so as to confirm the identity of the [3H]Ins(3,4,5,6)P4 peak that eluted from the HPLC. For these experiments, we did not assay [3H]inositol phosphates by on-line scintillation counting. Instead, individual HPLC fractions were collected. The amount of material loaded onto the HPLC column was increased by culturing greater numbers of cells in larger wells (see "Experimental Procedures"). Thus, the quantity of [3H]Ins(3,4,5,6)P4 was larger than in the experiments described by Fig. 5. Aliquots of the putative [3H]Ins(3,4,5,6)P4 peak were incubated with the purified 1-kinase (see "Experimental Procedures"). The [3H]InsP5 formed, after the assays had been allowed to proceed to completion, was used to identify the amounts of [3H]Ins(3,4,5,6)P4 in the original cell extracts: 6964 ± 35 dpm from control cells, and 13,322 ± 131 dpm after treatment with D/L-Bt3Ins(1,3,4)P3/AM. These experiments confirm that levels of [3H]Ins(3,4,5,6)P4 in intact cells were elevated 2-fold by Ins(1,3,4)P3.

We next investigated if the treatment of AR4-2J cells with Bt3Ins(1,3,4)P3/AM elicited increases in levels of Ins(1,3,4)P3 and Ins(3,4,5,6)P4 that were physiologically relevant. For these experiments, we compared the effects of the cell-permeant analogue with those of receptor-dependent activation of PLC, using bombesin as the agonist. [3H]Inositol-labeled AR4-2J cells were treated for 20 min with 200 nM bombesin, or for 60 min with either 100 µM D-Bt3Ins(1,3,4)P3/AM, or vehicle. The cell-permeant derivative used in these experiments was from a batch that was different from that used in the experiments described above. This particular batch of the analogue was also enantiomerically pure, and therefore it was used at half the concentration of the D/L-Bt3Ins(1,3,4)P3/AM used in the experiments described above. In three experiments, 60-min treatment of AR4-2J cells with 100 µM D-Bt3Ins(1,3,4)P3/AM elevated [3H]Ins(3,4,5,6)P4 levels 1.9 ± 0.3-fold (Fig. 6), which is not significantly different from the results obtained with 200 µM D/L-Bt3Ins(1,3,4)P3/AM (Table III). The fact that these changes in Ins(3,4,5,6)P4 levels were within a physiologically relevant range was confirmed by comparison with the effects of bombesin, which led to a nearly 5-fold increase in [3H]Ins(3,4,5,6)P4 levels (Fig. 6).


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  		Fig. 6.   Effect of bombesin and D-Bt3 Ins(1,3,4)P3/AM upon levels of [3H]Ins(3,4,5,6)P4 and non-radiolabeled Ins(1,3,4)P3 in intact AR4-2J cells. [3H]Inositol-labeled AR4-2J cells were incubated with either (i) 200 nM bombesin (BBS) or vehicle for 20 min, or (ii) 100 µM D-Bt3Ins(1,3,4)P3/AM or vehicle for 60 min. The cells were quenched and the inositol phosphates resolved by HPLC, as described under "Experimental Procedures." The changes in levels of [3H]Ins(3,4,5,6)P4 (gray bars) and non-radiolabeled Ins(1,3,4)P3 (black bars) were then determined as described under "Experimental Procedures." Means and standard errors are from three experiments.

We also compared the effects of D-Bt3Ins(1,3,4)P3/AM and bombesin upon cellular Ins(1,3,4)P3 levels (see under "Experimental Procedures"). Stimulation of AR4-2J cells with bombesin elicited a 13-fold increase in levels of Ins(1,3,4)P3 (Fig. 6); treatment with D-Bt3Ins(1,3,4)P3/AM elevated Ins(1,3,4)P3 levels 2.7-fold, an effect that may therefore be considered to be within the physiologically relevant range (Fig. 6).

Effects of InsP2 isomers on Ins(3,4,5,6)P4 1-Kinase Activity-- There is another important issue that is relevant to our evaluation of the significance of Ins(1,3,4)P3 as an intracellular signal. The extent to which metabolites of Ins(1,3,4)P3 also inhibit the 1-kinase contributes to the efficiency with which the cell "switches off" the Ins(1,3,4)P3 signal. The major route of Ins(1,3,4)P3 metabolism in vivo is by its dephosphorylation, by separate 4- and 1-phosphatases, to Ins(1,3)P2 and Ins(3,4)P2, respectively (24-26). We therefore determined the potency with which these bisphosphate degradation products inhibited the 1-kinase, relative to Ins(1,3,4)P3. The IC50 for Ins(1,3,4)P3 was 0.17 µM (Table II and Fig. 4A). In contrast, both Ins(1,3)P2 and D/L-Ins(3,4)P2 did not significantly affect 1-kinase activity until their concentration exceeded 3 µM (Fig. 7A); therefore, these bisphosphates were at least 100-fold weaker inhibitors. Thus, dephosphorylation of Ins(1,3,4)P3 is a very effective off-switch for relieving inhibition of 1-kinase activity.


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  		Fig. 7.   Effects of InsP2 isomers and D/L-Ins(1,2,4)P3 upon Ins(3,4,5,6)P4 1-kinase activity. 1-Kinase activity was assayed as described under "Experimental Procedures," in the presence of the following non-radiolabeled inositol phosphates: D/L-Ins(3,4)P2 (panel A, open squares), Ins(1,3)P2 (panel A, triangles), Ins(1,4)P2 (panel A, circles), D/L-Ins(1,2,4)P3 (panel B). Enzyme activity in each case is presented as a percentage of activity in the absence of any inhibitor. Data are derived from three or four experiments, and vertical bars denote standard errors (omitted from the experiments with InsP2 isomers for the sake of clarity).

The product of 5-phosphatase attack upon Ins(1,4,5)P3, namely Ins(1,4)P2, was found to be a poor inhibitor of the 1-kinase (Fig. 7A). This is also an important observation that demonstrates that it specifically requires Ins(1,4,5)P3 metabolism through the 3-kinase pathway to yield an inhibitor of the Ins(3,4,5,6)P4 1-kinase. In addition, this result shows that all three phosphates of Ins(1,3,4)P3 contribute substantially to the specificity of inhibition of the 1-kinase.

The Contribution of the 2-Phosphate Group to Inhibition of the 1-Kinase-- We found that, in an appropriate context, the addition of a 2-phosphate to the inositol ring could make a positive contribution to inhibitory potency; this is illustrated by the observation that Ins(1,2,3)P3 was a more potent inhibitor of the 1-kinase (IC50 = 4.2 µM, Table II) than was Ins(1,3)P2 (IC50 > 30 µM, Fig. 7A). D/L-Ins(1,2,3,4)P4 was also a relatively potent 1-kinase inhibitor (IC50 = 1.6 µM, Table II). D- and/or L-Ins(1,2,3,4)P4 and Ins(1,2,3)P3 are found in mammalian cells, the latter at concentrations of up to 10 µM (15, 42, 43). These polyphosphates may be formed by dephosphorylation of InsP6 (15, 42, 43). Among the inositol phosphates that we tested that contain a 2-phosphate (Table II), D/L-Ins(1,2,4)P3 was the most potent inhibitor of the 1-kinase (IC50 = 0.7 µM, Fig. 7B). Neither D- nor L-Ins(1,2,4)P3 have been detected in mammalian cells, even under circumstances where, had they been present, they should have revealed themselves to some detailed structural analyses (43). As the inhibitory action of Ins(1,3,4)P3 upon the 1-kinase was so effectively imitated by D/L-Ins(1,2,4)P3 (Table II), the latter could be a useful new starting point for developing drugs that might intervene in the 1-kinase/1-phosphatase cycle with therapeutic benefit.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Two aspects of this study are of particular importance to improving our understanding of the role of Ins(1,3,4)P3 as an intracellular signal. First, by using a new, cell-permeant, bioactivatable form of Ins(1,3,4)P3, we have shown that Ins(1,3,4)P3 elevates levels of Ins(3,4,5,6)P4 inside intact cells, without Ins(1,3,4)P3 being metabolized to Ins(3,4,5,6)P4. Second, we have discovered that Ins(1,3,4)P3-mediated inhibition of the Ins(3,4,5,6)P4 1-kinase is very efficiently switched off by dephosphorylation of Ins(1,3,4)P3. Prior to this study, there had not been a consensus of opinion concerning the mechanism by which PLC activation is coupled to increases in Ins(3,4,5,6)P4 levels. This is an important issue to resolve, if we are to understand how the cell regulates Ins(3,4,5,6)P4-mediated inhibition of conductance through Ca2+-activated Cl- channels (6, 8). We (15) have previously suggested that Ins(3,4,5,6)P4 levels are controlled by PLC-initiated regulation of the activities of a Ins(1,3,4,5,6)P5 1-phosphatase/Ins(3,4,5,6)P4 1-kinase substrate cycle (Fig. 1). Others have argued that Ins(1,3,4)P3 is converted to Ins(3,4,5,6)P4 by the sequential actions of Ins(1,3,4)P3 6-kinase, Ins(1,3,4,6)P4 1-phosphatase, and Ins(3,4,6)P3 5-kinase (22, 23, 44). An increased metabolic flux from Ins(1,3,4)P3 to Ins(3,4,5,6)P4 could elevate Ins(3,4,5,6)P4 levels by mass action effects. D/L-Bt3Ins(1,3,4)P3/AM enabled us to test this idea.

D/L-Bt3Ins(1,3,4)P3/AM provided a means of delivering a pulse of non-radiolabeled Ins(1,3,4)P3 into intact [3H]inositol-labeled cells. If, as a consequence of this treatment, there had been a significant metabolic flux from Ins(1,3,4)P3 into the Ins(3,4,5,6)P4 pool, this would have been expected to decrease the [3H]label in this Ins(3,4,5,6)P4 pool by a pulse-chase effect (15). In fact, the opposite result was obtained; D/L-Bt3Ins(1,3,4)P3/AM elevated intracellular levels of [3H]Ins(3,4,5,6)P4 (Table III). Thus, for the first time, we have shown that Ins(1,3,4)P3 regulates levels of Ins(3,4,5,6)P4 inside cells, without Ins(1,3,4)P3 being metabolized to Ins(3,4,5,6)P4.

An Ins(1,3,4)P3-mediated inhibition of Ins(3,4,5,6)P4 1-kinase in intact cells would be expected to alter the poise of the substrate cycle that interconverts Ins(3,4,5,6)P4 with Ins(1,3,4,5,6)P5 (Fig. 1). There was not a statistically significant decrease in the levels of [3H]Ins(1,3,4,5,6)P5 after treatment with D/L-Bt3Ins(1,3,4)P3/AM (Table III), but this is not unexpected. The [3H]Ins(1,3,4,5,6)P5 pool is relatively large, and it is difficult to detect the small changes in its size that are sufficient to support a 2-fold increase in the size of the much smaller [3H]Ins(3,4,5,6)P4 pool.

Ins(1,3,4)P3 would seem to be particularly well suited to its task as an intracellular mediator that links Ins(3,4,5,6)P4 levels to changes in PLC activity, since cellular levels of Ins(1,3,4)P3 quite closely follow both the extent and the duration of PLC activation (45, 46). In addition, we have shown the relative ineffectiveness with which the 1-kinase is inhibited by both of the InsP2 products of Ins(1,3,4)P3 metabolism, namely Ins(1,3)P2 and Ins(3,4)P2 (Fig. 7). Thus, the dephosphorylation of Ins(1,3,4)P3 comprises an efficient signaling off-switch. In this context, it now seems more significant that both the 1- and 4-phosphatases that attack Ins(1,3,4)P3 may be regulated. This is indicated first by the amino acid sequence of the 4-phosphatase containing consensus motifs for phosphorylation by protein kinases (47). Second, and more directly, Ca2+ activates the 1-phosphatase (40).

As a result of this study, we are wiser as to the structural determinants of Ins(1,3,4)P3-mediated inhibition of the 1-kinase (Table II). We also made the intriguing observation that a 2-phosphate could increase potency of 1-kinase inhibition, depending upon which other phosphate groups were also present. A practical outcome of this increased information concerning specificity is that the non-physiological material, D/L-Ins(1,2,4)P3, proved to be a particularly potent 1-kinase inhibitor; this could be a productive starting point for the rational design of therapeutically useful drugs that might inhibit the Ins(3,4,5,6)P4 1-kinase in vivo. This provides an alternative to the approach of designing drugs that act at the site of action of Ins(3,4,5,6)P4 (27).

The very existence of the Ins(1,4,5)P3 3-kinase, but more so its complex regulation through cross-talk from other signaling pathways, are observations that have been used to bolster the teleological argument that Ins(1,3,4,5)P4 must be functionally significant (2). Indeed, there is a large body of evidence that Ins(1,3,4,5)P4 does indeed perform a valuable role inside cells (2). The 3-kinase also has the role of inactivating Ca2+ signaling by Ins(1,4,5)P3. Our new data assign additional significance to this metabolic pathway: control over the production of Ins(1,3,4)P3, which in turn regulates cellular levels of Ins(3,4,5,6)P4 (an inhibitor of Ca2+-activated Cl- channels) (8). The acknowledgment that the Ins(1,4,5)P3 3-kinase has several important roles provides us with a better appreciation of why so many cellular control processes converge on the regulation of this enzyme's activity (48).

    FOOTNOTES

* The work in Pohang was supported by the Korean Science and Engineering Foundation/Center for Biofunctional Molecules and Ministry of Education Grant BSRI 97-3437), studies at Bath were supported by Wellcome Trust Program Grant 045491 (to B. V. L. P), and work in Bremen was supported by Deutsche Forschungsgemeinschaft Schu 943/1-6 (to C. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 919-541-3308; Fax: 919-541-0559; E-mail: yang3{at}niehs.nih.gov.

    ABBREVIATIONS

The abbreviations used are: InsPn, inositol polyphosphate, where n is the number of phosphates (e.g. InsP3); PEG, polyethylene glycol; PLC, phospholipase C; HPLC, high pressure liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; CaM KII, calmodulin-dependent protein kinase; Bt3Ins(1,3,4)P3/AM, 2,5,6-tri-O-butyryl-myo-inositol 1,3,4-trisphosphate hexakis (acetoxymethyl) ester.

    REFERENCES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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