Properties of the inositol 3,4,5,6-tetrakisphosphate 1-kinase purified from rat liver. Regulation of enzyme activity by inositol 1,3,4-trisphosphate.

Inositol 3,4,5,6-tetrakisphosphate is a novel intracellular signal that regulates calcium-dependent chloride conductance (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). The molecular mechanisms that regulate the cellular levels of this signal are not characterized. To pursue this problem we have now studied the 1-kinase that deactivates inositol 3,4,5,6-tetrakisphosphate. The enzyme was purified from rat liver 1600-fold with a 1% yield. The native molecular mass was determined to be 46 kDa by gel filtration. The Km values for inositol 3,4,5,6-tetrakisphosphate and ATP were 0.3 and 10.6 μM, respectively. The kinase was unaffected by either protein kinase A or protein kinase C. Increases in Ca2+ concentration from 0.1 to 1-2 μM inhibited activity by 10-20%. Most importantly, inositol 1,3,4-trisphosphate was shown to be a potent (Ki = 0.2 μM), specific, and competitive inhibitor of the 1-kinase. Our new kinetic data show that typical receptor-dependent adjustments in cellular levels of inositol 1,3,4-trisphosphate provide a mechanism by which the concentration of inositol 3,4,5,6-tetrakisphosphate is dependent on changes in phospholipase C activity. These conclusions also provide a new perspective to our understanding of the physiological importance of the pathway of inositol phosphate turnover initiated by the inositol 1,4,5-trisphosphate 3-kinase.

The agonist-mediated activation of phospholipase C and the resulting increased rate of hydrolysis of phosphatidylinositol 4,5-bisphosphate releases Ins(1,4,5)P 3 , 1 which mobilizes cellular Ca 2ϩ stores (1). The fact that the pathway by which Ins(1,4,5)P 3 is metabolized is complex has led to the speculation that one or more of the many downstream metabolites might serve important cellular functions (2)(3)(4). Support for this opinion has come mainly from research into the actions of Ins(1,3,4,5)P 4 , the product of 3-kinase-directed phosphorylation of Ins(1,4,5)P 3 . As a result of many studies, there is now considerable evidence that Ins(1,3,4,5)P 4 augments Ins(1,4,5)P 3 -initiated Ca 2ϩ mobilization (5). Yet the search for other signaling functions of additional inositol polyphosphates continues at several laboratories.
Attention has come to focus on a somewhat distant metabolic relative of Ins(1,4,5)P 3 , namely Ins(3,4,5,6)P 4 . The latter was first identified in mammalian cells in 1988, at which time it was noted that its levels increased substantially upon receptormediated activation of phospholipase C (6,7). We subsequently proposed that Ins(3,4,5,6)P 4 was an "orphan" second messenger (8,9). The first indication of what the function of this messenger might be came from work by Barrett, Traynor-Kaplan, and colleagues with the T84 colonic epithelial cell line (10,11). They drew attention to a correlation during receptor activation between the bulk InsP 4 content of the cell and the uncoupling of the customary ability of Ca 2ϩ to stimulate Cl Ϫ secretion. This prompted a study in which we treated T84 cells with cellpermeant analogues of inositol phosphates (12). The results indicated that Ins(3,4,5,6)P 4 was the specific InsP 4 isomer responsible for inhibiting Ca 2ϩ -dependent Cl Ϫ secretion (12). We subsequently consolidated this idea by demonstrating that the Ca 2ϩ -dependent Cl Ϫ conductance was directly inhibited by Ins(3,4,5,6)P 4 when it was microinjected into the T84 cell (13). Ins(3,4,5,6)P 4 had a similar effect when injected into CFPAC-1 cells, an adrenal carcinoma cell line deficient in cAMP-activated Cl Ϫ transport (14). Moreover, the cloned calcium-activated chloride channel from bovine trachea has also been shown to be directly inhibited by Ins (3,4,5,6)P 4 (15). In all three of these electrophysiological studies (13)(14)(15)) the effects of Ins(3,4,5,6)P 4 were very specific to that isomer, and they could not be imitated by up to 10-fold higher concentrations of other inositol polyphosphates.
Thus, Ins(3,4,5,6)P 4 may now be considered to be an authentic intracellular signal with the potential to modulate many of the physiological processes that require chloride channel activity: salt and fluid secretion, pH balance, neurotransmission, osmoregulation, and volume-dependent metabolic effects (16 -19). This has increased the importance of understanding the molecular mechanisms that regulate the synthesis and metabolic inactivation of this inositol polyphosphate. A particularly important aspect of this problem is to establish how the activation of phospholipase C is coupled to changes in cellular levels of Ins(3,4,5,6)P 4 . We have proposed that this cannot be explained as a simple mass action effect (8,9). That is, we have argued that a receptor-mediated increase in Ins(3,4,5,6)P 4 concentration does not reflect a nonspecific accumulation of downstream metabolites of Ins(1,4,5)P 3 (8,9). Instead we have proposed that agonists intervene in the activities of the Ins(3,4,5,6)P 4 1-kinase/Ins(1,3,4,5,6)P 5 1-phosphatase substrate cycle (8,9). However, this hypothesis has developed very little in the absence of any significant progress in determining the mechanism for this proposed regulatory process. In no small part, this uncertainty is due to the fact that no laboratory has yet purified the 1-phosphatase, and what we know of the 1-kinase depends almost entirely on a single paper by Stephens et al. (7). In that study, the enzyme in a 100,000 ϫ g supernatant made from rat brain was enriched approximately 35-fold by ammonium sulfate precipitation and anion-exchange chromatography (7). No kinetic information emerged from that work.
Assay of Ins (3,4,5,6)P 4  The final preparations of enzyme were also assayed using the above methods except that the quench medium did not contain InsP 6 . In some cases, the free Ca 2ϩ concentration was adjusted using EGTA/Ca 2ϩ buffers (21).
The reaction mixture was quenched with 1 ml of ice-cold medium containing 1 mg/ml InsP 6 , 0.2 M ammonium formate, 0.1 M formic acid. The quenched reactions were diluted to 10 ml with water and chromatographed on Bio-Rad gravity-fed columns using AG 1-X8 ion-exchange resin. In some experiments, incubations were quenched with 40 l of 2 M perchloric acid plus 1 mg/ml InsP 6 , neutralized with Freon/octylamine, and chromatographed on HPLC using a Partisphere SAX column (12).
Purification of Ins (3,4,5,6)P 4 1-Kinase: Preparation of 100,000 ϫ g Supernatant-Livers obtained from 200 -250-g male Sprague-Dawley rats were perfused with ice-cold saline and frozen at Ϫ60°C until use (freezing did not impair enzyme stability). All subsequent procedures (which took 5 days) were conducted at 0 -4°C, and all the column chromatography procedures (except for the final Matrex Blue A stage) utilized a Pharmacia FPLC system. For each preparation, about 70 g of liver was homogenized (500 -1000 rpm, five up-and-down strokes) in 140 ml of medium consisting of 20 mM Bis-Tris (pH 7.0), 1 mM EGTA, 1 g/ml leupeptin, 1 g/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged for 1.5 h at 100,000 ϫ g, and the resultant supernatant was filtered (0.2 m).
Heparin-Agarose Chromatography-The filtrated supernatant was loaded at about 1 ml/min to a heparin-agarose column (3.2 ϫ 20 cm) that was equilibrated with 300 ml of Buffer A (20 mM Bis-Tris (pH 7.0), 1 mM EGTA, 1 g/ml leupeptin, 1 g/ml pepstatin A) at a flow rate of 2 ml/min. After washing with 80 ml of Buffer A at 1.5 ml/min, the column was eluted at 1.5 ml/min with a linear gradient of 2.5-5 mM pyrophosphate in Buffer A for 300 min followed by 50 mM pyrophosphate in Buffer A for 200 min at 2 ml/min.
Phenyl-Agarose Chromatography-Peak fractions of enzyme activity eluted from the heparin column were pooled, the concentration of NaCl was adjusted to 1 M, and the sample was loaded onto a phenyl-Sepharose column (2.2 ϫ 10 cm) previously equilibrated with 250 ml of Buffer B (1 M NaCl, 10 mM Bis-Tris (pH 7.0), 1 mM EGTA, 1 g/ml leupeptin, 1 g/ml pepstatin A, and 10 mM pyrophosphate). After washing with 200 ml of Buffer B, the remaining protein was eluted with Buffer C (Buffer B without NaCl). The flow rate was 1 ml/min throughout.
Mono Q Anion-exchange Chromatography-Peak fractions of enzyme activity eluted from the phenyl-Sepharose column were pooled and loaded onto a Mono Q HR10/10 column previously equilibrated with 30 ml of Buffer D (50 mM Bis-Tris (pH 7.0), 1 mM EGTA, 1 g/ml leupeptin, 1 g/ml pepstatin A). The column was then washed with 10 ml of Buffer D and eluted with a linear gradient of 100 -350 mM NaCl in Buffer D for 50 min and 1 M NaCl in Buffer D for another 50 min. The flow rate was 0.5 ml/min throughout.
Matrex Blue A Chromatography-Peak fractions of enzyme activity eluted from the Mono Q fractions were pooled and 1 mM MgSO 4 was added. Samples were then loaded onto a 0.5-ml Matrex Blue A column previously equilibrated with Buffer D containing 1 mM MgSO 4 . The flow-through was collected under gravity. Finally, 1 mg/ml bovine serum albumin and 20% glycerol were added, and the preparations were stored at Ϫ70°C until use. There was no significant loss of activity over a 6-month period.
Gel Filtration-The molecular weight of the partially purified Ins(3,4,5,6)P 4 1-kinase was determined by gel filtration. Purified enzyme was concentrated about 10-fold using an Amicon Centricon-10 concentrator and mixed with molecular weight standards from Bio-Rad: bovine ␥-globulin, chicken ovalbumin, equine myoglobin, and vitamin B 12  Assay of the Effects of Protein Kinase C and Protein Kinase A on the Ins(3,4,5,6)P 4 1-Kinase-A 0.4-g aliquot of the Ins(3,4,5,6)P 4 1-kinase preparation was incubated with 0.05 units of protein kinase C (Pierce, catalog no. 29536) or 50 units of protein kinase A (Pierce, catalog no. 29538) in a volume of 10 l using the manufacturer's assay kit. After 10 min of incubation at 37°C, the mixtures were assayed for 1-kinase activity.
The activities of protein kinases A and C were also determined as positive controls using colorimetric assays (Pierce, catalog no. 29517 for kinase C and catalog no. 29529 for kinase A). (3,4,5,6)P 4 1-Kinase-Kinase activity during purification was assayed in the presence of 2 M InsP 6 . This had no effect on the 1-kinase (data not shown) but was used to inhibit the Ins(1,3,4,5,6)P 5 3-phosphatase (22) that contaminated the kinase during the initial stages of purification. However, the InsP 6 was not included in experiments with the purified enzyme since this did not contain any Ins(1,3,4,5,6)P 5 phosphatase activity (data not shown).

Purification of Ins
We chose to isolate the 1-kinase from liver, a tissue in which the enzyme has relatively high specific activity (7); kinetic data we accumulated from the purified enzyme could than be compared with the inositol phosphate profiles in isolated hepatocytes (23). The rat hepatic 1-kinase is predominantly a soluble enzyme (Ref. 7 and data not shown). These crude preparations of enzyme were relatively stable when maintained at 0 -4°C, but our initial attempts at further purification were thwarted by such procedures bringing about a nearly complete loss of activity. For example, if the enzyme was concentrated by ammonium sulfate precipitation, 98% of activity was lost within 48 h (Table I). Fortunately, we discovered that certain phosphate-containing compounds and some phosphatase inhibitors were able to preserve enzyme activity to varying extents (Table  I). Among them, inorganic pyrophosphate was the most effective. The disadvantage of including pyrophosphate was that it prevented the enzyme from binding to many of the column resins that otherwise might have facilitated the purification of the enzyme. Eventually, we found that an adequate compromise was to load the crude supernatant onto a heparin-agarose column in the absence of pyrophosphate and then use this compound to elute the enzyme from the column as a single peak (Fig. 1A, Table II). These procedures brought about an approximately 20-fold purification with an apparent yield of 67%.
Pyrophosphate was also present during the next stage of purification of the enzyme by phenyl-Sepharose chromatography (Fig. 1B , Table II). However, during further purification steps (Fig. 1C, Table II) the enzyme again became unstable at 0 -4°C, apparently for a different reason since pyrophosphate now offered no protective effect (a fact that also prevented us from using the purified enzyme to explore the mechanism of action of pyrophosphate). We did discover that enzyme activity could be preserved by the addition of excess protein (e.g. 1 mg/ml bovine serum albumin). It is possible that the enzyme was now being adversely affected by its inevitable dilution during purification. Neither glycerol (up to 50% (v/v)) nor polyvinylpyrrolidone (up to 0.5% (w/v)) were effective substitutes for the albumin. We attempted to compensate for this particular difficulty by scaling up our preparations to increase the concentration of the purified 1-kinase. We were then able to further purify the enzyme by anion-exchange chromatography (Fig. 1C, Table II) and Matrex Blue A dye-ligand chromatography (Table II). The final preparations, which were approximately 1600-fold pure with a 1% yield, were stored at Ϫ70°C with the addition of 1 mg/ml bovine serum albumin plus 20% (v/v) glycerol; there was no significant loss of activity for at least 6 months.
Properties of the 1-Kinase-We found that the enzyme had high affinity for both Ins(3,4,5,6)P 4 and ATP; the K m value of the 1-kinase for Ins(3,4,5,6)P 4 was estimated to be 0.36 Ϯ 0.01 M (mean Ϯ S.E. from three determinations, Fig. 2A). The kinetic properties of the 1-kinase activity in any tissue have not previously been described. Our determinations were made possible by the development of procedures for the chemical synthesis of enantiomerically pure Ins(3,4,5,6)P 4 (13)
Regulation of 1-Kinase Activity-The most important goal of this study was to gain insight into how the 1-kinase might be regulated in vivo, particularly when phospholipase C is activated. We found that the 1-kinase activity was unaffected by its co-incubation with either protein kinase A or protein kinase C (data not shown; for details, see "Experimental Procedures"). We also investigated the effect on the 1-kinase of the changes in [Ca 2ϩ ] that would be expected to occur in cells upon activation of phospholipase C. The enzyme activity was slightly reduced (ϳ10 -20%) at 1-2 M Ca 2ϩ relative to the activity at 0.1 M (data not shown). This small effect, which would only be expected to occur during the brief period at which a cellular Ca 2ϩ transient attained its peak value, was not influenced (data not shown) by the further addition of either 5 M calmodulin or 10 M calmodulin antagonist (kinase fragment 290 -309 (24)). From the point of view of our efforts to understand how the Ins(3,4,5,6)P 4 1-kinase is regulated, a particularly important result (see "Discussion") was the potency with which this enzyme was inhibited by Ins(1,3,4)P 3 (Fig. 4); the inhibition was determined by a Dixon plot to be competitive (Fig. 4), and the estimated K i value was 0.2 Ϯ 0.01 M (mean Ϯ S.E., n ϭ 3; Fig.  4, Table III). The inhibition of 1-kinase by Ins(1,3,4)P 3 was relatively specific; Ins(1,4,5)P 3 , Ins(1,3,4,5)P 4 , Ins(1,3,4,6)P 4 , and Ins(1,3,4,5,6)P 5 were also competitive inhibitors, but they all acted with considerably lower potency (Table III). DISCUSSION In order to fully discern the actions of second messengers we need to determine how both their accumulation and metabolic inactivation are regulated. This generally requires the purification and characterization of the enzymes responsible for these metabolic control processes. This is the approach we have taken to increase our understanding of the receptor-regulated mechanisms that control metabolic turnover of Ins(3,4,5,6)P 4 , a novel cellular signal (see the Introduction). Thus, we have purified the Ins(3,4,5,6)P 4 1-kinase activity 1600-fold from a hepatic supernatant with a 1% yield. (The only previously described purification of this enzyme yielded a 35-fold enriched sample from a rat brain supernatant (7).) The novel kinetic data that we have obtained with this purified enzyme have been particularly revealing concerning the mechanisms by which receptor-mediated activation of phospholipase C causes cellular Ins(3,4,5,6)P 4 levels to increase. For example, we have discovered that the 1-kinase has relatively high affinity for Ins(3,4,5,6)P 4 (K m ϭ 0.36 M). In addition, we have determined that Ins(1,3,4)P 3 is a potent inhibitor of the 1-kinase, with a K i of 0.2 M. Since intracellular levels of Ins(1,3,4)P 3 are about 1 M under basal conditions (25), inhibition of the 1-kinase by this polyphosphate is physiologically relevant. Inhibition by Ins(1,3,4)P 3 was also relatively specific; 2 Several bands were observed when the enzyme preparations were analyzed by SDS-polyacrylamide gel electrophoresis and silver-stained; one of these migrated with an apparent molecular mass of 46 kDa. Densitometric analysis indicated that this particular band accounted for approximately 15% of total protein (data not shown).

TABLE I
Enzyme stability under different conditions Ins(3,4,5,6)P 4 1-kinase activity in 100,000 ϫ g supernatant was precipitated by 50% (w/v) ammonium sulfate, resuspended, and dialyzed overnight in homogenization medium. Aliquots were incubated with the indicated reagent and assayed either immediately or after 2 days at 4°C. Reactions were quenched and analyzed as described under "Experimental Procedures." other inositol polyphosphates were inhibitors but at unphysiologically high concentrations (Table III), with perhaps one exception. In intact cells some slight inhibition of 1-kinase activity by Ins(1,3,4,5,6)P 5 is also possible since its steady-state levels (22,23) are close to its K i value of 15 M. However, levels of this particular isomer do not change substantially during activation of phospholipase C (4,9), so this polyphosphate will not contribute to receptor-mediated regulation of the 1-kinase. It is of further significance that Ins(1,3,4)P 3 is a downstream metabolite of Ins(1,4,5)P 3 (4). Thus, the intracellular levels of Ins(1,3,4)P 3 accumulate severalfold whenever phospholipase C is activated. This raises the possibility that it is these receptor-mediated changes in Ins(1,3,4)P 3 levels that, by inhibiting 1-kinase, link phospholipase C activity to elevations in Ins(3,4,5,6)P 4 levels. To further explore this idea, we fitted our kinetic data to Equation 1 (see Ref. 22).
Therefore, we conclude that a major function of Ins(1,3,4)P 3 is to provide a mechanism by which activation of phospholipase C can induce changes in cellular levels of Ins(3,4,5,6)P 4 . In this way phospholipase C may be considered to have both positive and negative input into Cl Ϫ ion channel activity: initial stimulation by Ca 2ϩ followed by inhibition by Ins(3,4,5,6)P 4 . The  idea that Ins(1,3,4)P 3 serves an important physiological function amends a long-standing belief that it is merely an inactive degradation product of Ins(1,4,5)P 3 /Ins(1,3,4,5)P 4 turnover. Moreover, this role for Ins(1,3,4)P 3 is ultimately served by the activity of the Ins(1,4,5)P 3 3-kinase. Thus, both the function of the 3-kinase and the selective pressures that caused it to evolve (see Ref. 5) should no longer be interpreted purely in terms of regulating the levels of Ins(1,4,5)P 3 and Ins(1,3,4,5)P 4 to control Ca 2ϩ mobilization. It is entirely possible that this action of Ins(1,3,4)P 3 by itself is all that is necessary to elevate levels of Ins(3,4,5,6)P 4 in vivo.
In any case, the identification of this process adds a remarkable new feature to our understanding of the control of signaling functions of inositol polyphosphates. These results also consolidate our original hypothesis (8,9) that receptor-dependent changes in Ins(3,4,5,6)P 4 levels are not just a consequence of mass action effects dependent on alterations in levels of its metabolic precursor, i.e. Ins(1,3,4,5,6)P 5 . Moreover, our data exclude a substantial contribution of some other potential regulatory processes to the control of 1-kinase. For example, the enzyme activity was not affected by either protein kinase A or protein kinase C. We also investigated whether the changes in cellular [Ca 2ϩ ] that occur upon receptor activation would affect 1-kinase activity. When free [Ca 2ϩ ] in our assays was raised from 0.1 to 1-2 M, there was a slight inhibition of enzyme activity (10 -20%), but this seems insufficient to substantially contribute to the control of 1-kinase activity in vivo. Indeed, we have previously shown that Ins(3,4,5,6)P 4 levels in primary cultures of rat hepatocytes did not change in response to thapsigargin (which elevates cytosol [Ca 2ϩ ]). The high affinity of the kinase for ATP (K m ϭ 10.6 M) indicates that the activity of the hepatic enzyme in vivo is unlikely to be affected by physiologically relevant changes in ATP levels, which are maintained in the millimolar range (26).
Drugs that modify the actions and metabolism of Ins(3,4,5,6)P 4 might improve treatment for certain medical conditions that result from perturbations to transmembrane Cl Ϫ transport, such as cystic fibrosis, and heart arrhythmia. The new information we have provided concerning the properties of the 1-kinase may improve the scope for pharmacological intervention, since they identify the enzymes of Ins(1,3,4)P 3 metabolism as additional, potential therapeutic targets. Finally, our goal of sequencing and cloning the 1-kinase will help us understand the properties of this enzyme at a molecular level.