©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphatidylinositol 4-Phosphate Increases the Rate of Dephosphorylation of the Phosphorylated Ca-ATPase (*)

Anthony P. Starling , J. Malcolm East , Anthony G. Lee

From the (1)Department of Biochemistry, University of Southampton, Southampton SO9 3TU, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Incubation of the Ca-ATPase of skeletal muscle sarcoplasmic reticulum with ATP in the absence of Ca leads to phosphorylation of phosphatidylinositol (PtdIns) to phosphatidylinositol 4-phosphate (PtdIns-4P) and to a doubling of ATPase activity. Similarly, reconstitution of the ATPase with mixtures of dioleoylphosphatidylcholine and PtdIns-4P also led to a doubling of activity; ATPase activity increased with increasing PtdIns-4P content, up to 10% beyond which no further increase was observed. Reconstitution with PtdIns had a much smaller effect on activity. Changes in the Ca affinity of the ATPase following incubation with ATP or reconstitution with PtdIns-4P were small. The rates of phosphorylation of the ATPase by ATP and of the Ca transport step were unaffected, but the rate of dephosphorylation of the phosphorylated ATPase increased by a factor of 2 either following incubation with ATP or following reconstitution with PtdIns-4P. Activation of the ATPase led to a decrease in the level of phosphorylation of the ATPase by P corresponding to a 10-fold decrease in the equilibrium constant E2PMg/E2PMg.


INTRODUCTION

The major phospholipid in the sarcoplasmic reticulum (SR)()of skeletal muscle is phosphatidylcholine(1, 2) . Phosphatidylcholines have been shown to interact nonspecifically with the hydrophobic membrane-penetrant parts of the Ca-ATPase of SR and are thus believed to provide the bulk ``solvent'' lipids for the ATPase(1, 3) . For the ATPase to be active, these lipids must be in the liquid crystalline phase(4, 5) , and optimal activity is obtained with a chain length of C18, that is, with dioleoylphosphatidylcholine (di(C18:1)PC)(1, 6, 7, 8) .

As well as these solvent-like lipids, a few ``special'' lipid molecules may bind at distinct sites on the ATPase. SR membranes contain, as well as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol (PtdIns), and phosphatidylinositol 4-phosphate (PtdIns-4P), but no phosphatidylinositol 4,5-bisphosphate(2) . The absence of phosphatidylinositol 4,5-bisphosphate means that, unlike the transverse tubule membrane, SR lacks the capacity for Ins(1,4,5)P generation(2) . PtdIns 4-kinase has been detected in SR, as has a Ca-dependent phosphomonoesterase capable of catalyzing the breakdown of PtdIns-4P(9, 10) . A possible physiological role for PtdIns-4P is suggested by the experiments of Varsanyi et al.(11) who showed that incubation of the purified ATPase with ATP in the absence of Ca led to phosphorylation of up to one PtdIns molecule per ATPase molecule to form PtdIns-4P and that this resulted in stimulation of ATPase activity. Here, we show that the effect of PtdIns-4P on ATPase activity follows from a specific effect on the rate of dephosphorylation of the phosphorylated ATPase.


EXPERIMENTAL PROCEDURES

Materials

Dioleoylphosphatidylcholine was purchased from Avanti Polar Lipids, Inc. (Birmingham, AL), and PtdIns and PtdIns-4P were from Sigma. Sarcoplasmic reticulum and purified Ca-ATPase were prepared from rabbit skeletal muscle(3) . Concentrations of ATPase were estimated by using the extinction coefficient (1.2 liters g cm for a solution in 1% SDS) given by Hardwicke and Green(12) .

Reconstitution with PtdIns-4P

Mixtures of di(C18:1)PC and PtdIns or PtdIns-4P (10 µmol, total phospholipid) in buffer (400 µl, 10 mM Hepes/Tris, pH 8.0, containing 15% sucrose, 5 mM MgSO, 5 mM ATP, and 12 mg/ml potassium cholate) were sonicated to clarity in a bath sonicator. ATPase (1.25 mg) in a volume of 20-30 µl was then added, and the mixture was left for 15 min at room temperature and 45 min at 5 °C to equilibrate. After equilibration, the samples were added to precooled Oakridge tubes containing ice-cold buffer (10 mM Hepes/Tris, pH 8.0, 2 mM dithiothreitol) and centrifuged at 200,000 g for 1 h at 4 °C. Samples were rehomogenized and suspended in buffer (10 mM Hepes/Tris, 15% sucrose) to a concentration of 3-8 mg/ml and then stored at -20 °C until use.

Activation with ATP

The purified Ca-ATPase or SR vesicles were activated by incubation with ATP largely as described by Varsanyi et al.(11) . The ATPase or SR vesicles (2 mg of protein/ml) were incubated with 1 mM ATP in 40 mM Hepes/KOH, pH 7.2, containing 100 mM KCl, 5 mM MgSO, and 1 mM EGTA for 1 h at 25 °C. The sample was then centrifuged at 100,000 g for 1 h, and the pellet was kept on ice until use. As a control, ATPase was incubated under the same conditions but in the absence of ATP.

ATPase Assay

ATPase activities were determined at 25 °C by using a coupled enzyme assay in a medium containing 40 mM Hepes/KOH, pH 7.2, 100 mM KCl, 5 mM MgSO, 2.1 mM ATP, 1.1 mM EGTA, 0.41 mM phosphoenolpyruvate, 0.15 mM NADH, pyruvate kinase (7.5 IU), and lactate dehydrogenase (18 IU) in a total volume of 2.5 ml. The reaction was initiated by addition of an aliquot of a 25 mM CaCl solution to a cuvette containing the ATPase and the other reagents to give the required concentration of Ca. Free concentrations of Ca were calculated using the binding constants for Ca, Mg, and H to EGTA given by Godt(13) .

Rapid Kinetic Experiments

The time dependence of phosphorylation-induced Ca release from the ATPase was determined using a Biologic Rapid filtration system, and the time dependences of phosphorylation of the ATPase by [-P]ATP and of dephosphorylation of the ATPase phosphorylated with [P]P in the absence of Ca, or with [-P]ATP in the presence of Ca, were determined using a Biologic QFM-5 system as described by Starling et al.(14) .

Equilibrium Levels of Phosphorylation with P

The ATPase (0.2 mg of protein/ml) was incubated with [P]P in 150 mM Mes/Tris, pH 6.2, containing 5 mM EGTA and the required concentrations of Mg at 25 °C. After 15 s, the reaction was quenched by addition of 10 volumes of quenching solution (25% trichloroacetic acid, 0.13 M phosphoric acid). The sample was put on ice for 15 min, and then the precipitate was collected by filtration through Whatman GF/B glass fiber filters and finally counted in Optiphase Hisafe III.

Phosphorylation of PtdIns by ATP

SR vesicles (25 mg of protein) were incubated with ATP as described above, followed by precipitation with trichloroacetic acid. The precipitate was pelleted, washed with water, dried in vacuo, and extracted with chloroform/methanol (3 ml; 2:1 v/v). The precipitate was again pelleted and then extracted with chloroform/methanol/HCl (3 ml, 40:20:1 (v/v/v)). The organic layer was filtered through a Whatman GF/C filter and washed with 0.6 ml of a 1% NaCl solution, and the lower chloroform-rich phase evaporated in vacuo. Lipids were separated by thin layer chromatography on Silica gel 60 plates using chloroform/methanol/water/ammonia (48:40:7:5 (v/v/v/v)) as solvent (15). Lipids were visualized by staining with iodine and P-labeled lipid detected using a plate scanner (Dünnschicht).


RESULTS

ATPase Activity

If the ATPase is incubated with MgATP in the absence of Ca for 1 h and then the rate of ATP hydrolysis measured at 100 µM Ca, an activity of 5.9 IU/mg protein is obtained, compared with a value of 3.0 IU/mg protein for the ATPase prior to incubation with ATP (Fig. 1).


Figure 1: ATPase activity for the activated or reconstituted ATPase. Shown are the ATPase activities (IU/mg protein) for the ATPase with () or without () activation in the presence of ATP and for the ATPase reconstituted with an 8:1 molar ratio of di(C18:1)PC or PtdIns-4P () or PtdIns (). ATPase activities were measured at ph 7.2, 2.1 mM ATP, and the given concentration of Ca, at 25 °C.



The ATPase was reconstituted into bilayers of di(C18:1)PC and PtdIns-4P by mixing the ATPase with lipid in cholate solution at a molar ratio of lipid:ATPase of 1000:1, followed by dilution into buffer to reform membrane fragments(1) . Following reconstitution with a lipid mixture containing 20% PtdIns-4P, the ATPase activity was the same as that for the ATPase activated with ATP (Fig. 1). The effect of PtdIns-4P on ATPase activity increased with increasing PtdIns-4P content up to 10%, beyond which activity was constant up to 50% PtdIns-4P, the highest content tested (data not shown). Reconstitution with mixtures of di(C18:1)PC and PtdIns led to smaller increases in ATPase activity (Fig. 1), with stimulation of ATPase activity reaching a maximum at 10% PtdIns.

The dependence of ATPase activity on Ca concentration is complex (Fig. 1) with low concentrations of Ca activating the ATPase, attributable to binding of Ca to the Ca binding sites on the unphosphorylated ATPase (E1), and high concentrations of Ca inhibiting the ATPase, attributable both to binding of Ca to the phosphorylated ATPase (E2P) with a subsequent decrease in the rate of dephosphorylation and to the formation of CaATP, which is hydrolyzed more slowly by the ATPase than MgATP(16, 17, 18) . Activation with ATP or reconstitution with PtdIns-4P had no significant effect on the Ca dependence of activity, in either the low or high concentration regions (Fig. 1). The affinity of the ATPase for Ca can also be measured from changes in the tryptophan fluorescence intensity of the ATPase on binding Ca(19) . Such measurements detect a very small decrease in the Ca affinity (a shift in the pCa value for 50% binding of -0.2) on reconstitution with PtdIns-4P (data not shown).

The Rate of Phosphorylation of the ATPase

Mixing the ATPase incubated in the presence of 100 µM Ca with 50 µM [-P]ATP at pH 7.2 leads to rapid formation of phosphoenzyme, which fits to a single exponential process with rates of 78.7 ± 10.0 s and 77.6 ± 9.2 s, respectively, for the ATPase before and after activation by incubation with ATP (data not shown). The maximum level of phosphorylation, 2.8 nmol of [EP]/mg protein, was unaffected by incubation with ATP, corresponding to a fraction of active protein of about 0.3, typical for this and other preparations of the ATPase.

The Rate of Phosphorylation-induced Ca Release

The rate of phosphorylation of the ATPase by ATP in the presence of Ca is much faster than the rate of dissociation of Ca from the unphosphorylated ATPase(20) . Under these conditions, Orlowski and Champeil (20) have shown that the rate of Ca dissociation from the phosphorylated ATPase (the E1PCa E2P step) can be measured by pre-equilibrating the ATPase with Ca and then perfusing it on Millipore filters with Ca and ATP. When the ATPase was incubated with 100 µMCa in buffer at pH 7.2 and then perfused with the same medium containing 100 µM unlabeled Ca and 2 mM MgATP, dissociation of Ca from the ATPase was observed, fitting to a single exponential process with a rate constant of 19.3 ± 4.6 (Fig. 2). If the ATPase was first activated by incubation with ATP in the absence of Ca for 1 h, then the rate of Ca dissociation was not changed significantly, fitting to a rate constant of 15.5 ± 3.3 s (Fig. 2).


Figure 2: ATP-induced release of Ca from the ATPase. The ATPase (0.4 mg/ml) with () or without () activation with ATP was equilibrated in buffer (20 mM Hepes/Tris, pH 7.2, 100 mM KCl, 20 mM Mg) containing 100 µMCa and 0.5 mM [H]sucrose, and then 0.1 mg of ATPase was adsorbed onto Millipore 0.45-µm HAWP filters. The loaded filter was perfused for the given times with the same buffer containing 100 µM unlabeled Ca and 2 mM ATP, and the amount of Ca bound to the ATPase was determined (14). The solidline shows a single exponential fit to the data for the unactivated ATPase, with a rate constant of 19.3 ± 4.5 s.



Phosphorylation of the ATPase by P

Incubation of the ATPase with P at pH 6.2 in the presence of Mg and absence of Ca leads to phosphorylation of the ATPase. The level of phosphorylation observed is reduced if the ATPase is first activated by incubation with ATP (Fig. 3). Phosphoenzyme formation fits to Fig. SI(21, 22) .


Figure 3: Phosphorylation of the ATPase by P. The ATPase (0.2 mg/ml) was incubated in 150 mM Mes/Tris, pH 6.2, 5 mM EGTA, and 10 mM Mg in the presence of the given concentration of phosphate (A) and 1 mM P and the given concentration of Mg:ATPase with () and without () activation with ATP (B). The solidlines show simulations calculated as described in the text assuming a value of [EP] of 3.5 nmol/mg protein.




Figure SI: Scheme I.



As described(22) , good fits to the experimental data for the unstimulated ATPase can be obtained with binding constants of Mg and P of 100 M, with an equilibrium constant E2PMg/E2PMg of 18 and an equilibrium constant E1/E2 calculated as described by Henderson et al.(19) (Fig. 3). For the activated ATPase, the data fit to the same binding constants for Mg and P but with a reduced value for the equilibrium constant E2PMg/E2PMg of 1.8 (Fig. 3).

Rate of Dephosphorylation of the ATPase

The rate of dephosphorylation of the phosphorylated ATPase can be determined either by phosphorylating the ATPase with [P]P at pH 6.0 in the absence of Ca and presence of dimethyl sulfoxide followed by mixing with an excess of a pH 7.5 buffer containing KCl and ATP to induce dephosphorylation, or by first phosphorylating the ATPase with [-P]ATP in the presence of Ca and then mixing with an excess of unlabeled ATP(14) . Although the level of phosphorylation of the activated ATPase by P is normally low (Fig. 3), in the presence of 14% (v/v) dimethyl sulfoxide, levels of phosphorylation are comparable for the activated and the non-activated ATPase. Dephosphorylation of the ATPase phosphorylated with P in the presence of dimethyl sulfoxide fits to a single exponential process with a rate of 15.2 ± 1.6 s before activation of the ATPase with ATP and 36.9 ± 3.0 s after activation with ATP (Fig. 4A). A similar increase in the rate of dephosphorylation was observed following reconstitution with PtdIns-4P. Rates of dephosphorylation have been observed to vary between preparations of the ATPase, and for that used to obtain the data in Fig. 4B, a rate of dephosphorylation of 7.6 ± 0.6 s was observed when reconstituted in di(C18:1)PC, increasing to 15.1 ± 2.0 s on reconstitution with a mixture of di(C18:1)PC and PtdIns-4P at a molar ratio of 8:2 (Fig. 4B). For the ATPase phosphorylated with ATP, rates of dephosphorylation of 11.6 ± 2.5 s and 26.8 ± 5.9 s were observed before and after activation with ATP, respectively (Fig. 4C).


Figure 4: The rate of dephosphorylation of the phosphorylated ATPase. A and B, the enzyme syringe contained ATPase (4 mg/ml) in 12.5 mM Mes/Tris, pH 6.0, containing 10 mM EGTA, 1 mM [P]P, 20 mM Mg, and 14% (v/v) dimethyl sulfoxide. The second syringe contained 100 mM Mes/Tris, pH 7.5, containing 100 mM KCl, 4 mM Mg, and 5.3 mM ATP. The contents of the enzyme syringe were mixed in a 1:16 volume ratio with the dephosphorylation mixture and the reaction quenched at the given times with 25% trichloroacetic acid. A, ATPase with () and without () activation with ATP. B, ATPase reconstituted with di(C18:1)PC () or with an 8:2 molar ratio of di(C18:1)PC and PtdIns-4P (). C, the enzyme syringe contained ATPase (0.2 mg/ml) in 20 mM Mes/Tris, pH 7.2, 5 mM Mg, 100 mM KCl, and 100 µM Ca. This was mixed in a 1:1 ratio with a solution containing 50 µM [-P]ATP in the same buffer. The mixture was incubated for 200 ms and then mixed in a 1:1 ratio with the same buffer containing 2.5 mM unlabeled ATP. The reaction was quenched at the given time with 25% trichloroacetic acid. ATPase with () and without () activation with ATP is shown. The solidlines show fits to single exponential processes, with the rate constants given in the text.



Phosphorylation of PtdIns by ATP

As described by Varsanyi et al.(11) , incubation of SR vesicles with [-P]ATP in the absence of Ca led to the formation of radiolabeled PtdIns-4P, with no detectable radiolabel incorporation into phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine.


DISCUSSION

The activity of the Ca-ATPase in cardiac sarcoplasmic reticulum is modified by interaction with phospholamban. Binding of phospholamban to the Ca-ATPase reduces the maximal rate of ATP hydrolysis and reduces the affinity of the ATPase for Ca(23, 24) , the hydrophilic domain of phospholamban reducing v by reducing the rate of the Ca transport step(25) . No such control mechanism has yet been established for the Ca-ATPase in skeletal muscle sarcoplasmic reticulum.

Skeletal muscle SR shows the kinase activity necessary to phosphorylate PtdIns to PtdIns-4P(2, 10, 11) . It also contains a Ca-dependent phosphomonoesterase able to hydrolyze PtdIns-4P(10) . Varsanyi et al.(11) have shown that conversion of a small number of the PtdIns molecules in the SR membrane to PtdIns-4P (probably one per ATPase molecule) leads to stimulation of the Ca-ATPase(11) . The PtdIns-4P involved in stimulation of the ATPase is probably tightly bound to it, since it is insensitive to a variety of phospholipases(11) .

We have confirmed these results. Incubation of the Ca-ATPase with ATP in the absence of Ca leads to phosphorylation of PtdIns to PtdIns-4P, this resulting in stimulation of the ATPase (Fig. 1). The affinity of the ATPase for Ca is little changed (Fig. 1), confirmed by measurements of changes in tryptophan fluorescence intensity as a function of Ca concentration (data not shown). Stimulation of the ATPase can also be achieved by reconstitution of the ATPase with mixtures of di(C18:1)PC and PtdIns-4P (Fig. 1). The high molar ratio of PtdIns-4P to ATPase required for maximal stimulation in the reconstituted system (100:1) suggests significant partitioning of PtdIns-4P between the site(s) on the ATPase and the bulk lipid phase.

In previous studies of the effects of phospholipids on the activity of the ATPase, we have detected changes in the rates of phosphorylation and dephosphorylation of the ATPase(5, 8) , and stimulation of the ATPase by jasmone has been shown to follow from increases in the rates of the Ca transport step and of dephosphorylation(14) . Effects of jasmone and activation of the ATPase by ATP are additive; addition of 200 µM jasmone increases the activity of the activated ATPase from 5.9 to 7.9 IU/mg protein, measured under the conditions shown in Fig. 1(data not shown).

Activation of the ATPase with ATP has no effect on the rate of phosphorylation of the ATPase (data not shown) or on the rate of the Ca transport step (Fig. 2). However, it does have a marked effect on the rate of dephosphorylation of the phosphorylated ATPase, this increasing by a factor of about 2 (Fig. 4). The rate of dephosphorylation of the ATPase in a mixture of di(C18:1)PC and PtdIns-4P is also found to be double that for the ATPase in di(C18:1)PC (Fig. 4). Equilibrium measurements of phosphorylation of the ATPase by P (Fig. 3) are consistent with a 10-fold decrease in the equilibrium constant for phosphorylation (E2PMg/E2PMg, Fig. SI) on activation with ATP. With a doubling of the rate of dephosphorylation, this implies a 5-fold decrease in the rate of phosphorylation by P on activation of the ATPase. Since the binding site for PtdIns-4P is presumably in the trans-membrane region of the ATPase, the specific effect of PtdIns-4P on the E2PMg&rlarr2;E2PMg step implies a long range interaction on the ATPase, since the phosphorylation domain of the ATPase is located a considerable distance above the membrane surface(26) .

PtdIns-4P and other negatively charged phospholipids have been shown to increase the affinity of the plasma membrane Ca-ATPase for Ca but with no effect on v(27) . The binding site for PtdIns-4P on the plasma membrane Ca-ATPase has been suggested to involve both a large positively charged loop just before the third transmembrane -helix and the C-terminal calmodulin binding domain (27). Although the N-terminal region of the first of these proposed sites is absent from the SR Ca-ATPase, the C-terminal region, corresponding to the sequence DKTPLQQKLDEFGE in the SR Ca-ATPase, containing two conserved Lys residues is present as part of the proposed third stalk region(28) . This region could therefore be involved in binding PtdIns-4P to the SR Ca-ATPase. Alternatively, the binding site for PtdIns-4P on the SR ATPase could be in the distinct central space between trans-membrane lobes A, B, and C seen in electron micrographs(29, 30) .


FOOTNOTES

*
This work was financially supported in part by the BBSRC. 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.

The abbreviations used are: SR, sarcoplasmic reticulum; di(C18:1)PC, dioleoylphosphatidylcholine; PtdIns, phosphatidylinositol; PtdIns-4P, phosphatidylinositol 4-phosphate; Mes, 4-morpholine-ethanesulfonic acid.


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