In vivo regulation of phosphorylation level and activity of phosphofructokinase by serotonin in Fasciola hepatica

AKCHIVES OF RIOCHEMISTRY ANL) BIOPHYSICS Vol. 271, No. 2, June, pp. 553-559, 1989

In Vivo Regulation Phosphofructokinase EDWIN

of Phosphorylation by Serotonin

S. KAMEMOTO,

LINDA

Received

Level and Activity of in Fasciola hepatica’52

LAN,

December

AND

TAG E. MANSOUR

14,1988

The level of phosphorylation and activation of phosphofructokinasc by serotonin (5 hydroxytryptamine) was studied in intact liver flukes Fasciola hepatica. The enzyme was immunoprecipitated with antiserum prepared against pure enzyme from the liver flukes. The resuspended immunoprecipitated enzyme retained most of its original activity and its kinetic properties. The level of phosphorylation was determined by a “back phosphorylation” technique. According to this technique, the immunoprecipitated phosphofructokinase was phosphorylated with the catalytic subunit of pure CAMP-dependent protein kinase. Incubation of intact liver flukes with serotonin caused an increase in the level of enzyme phosphorylation which was concomitant with an increase in enzyme activity. The level of phosphorylation was increased by 0.08 mol per protomer as a result of maximal activation by serotonin. It is proposed that phosphorylation plays, at least in part, a functional role in the regulation of phosphofructokinase from the liver fluke F. h,eprr,tica under in +?o conditions. (it 1YRY Academic Prey, Inr

The liver fluke Fasciola hepaticu, is a trematode that lives in the bile ducts of cattle, sheep, and occasionally in man. It depends for its survival on anaerobic glycolysis. Serotonin (5-hydroxytryptamine) receptors that are linked to adenylate cyclase are present in the parasite (1). It was previously shown that serotonin increases the rate of glycolysis in intact organisms as well as in cell free extracts (2, 3). The indolamine appears to have the same effects on the parasite as that of epinephrine in the mammalian skeletal muscle, i.e., activation of adenylate cyclase, cyclic AMP-dependent protein kinase, and phos’ This work was supported of Health Grant. AI 16501. Consortium on the Biology ported by the John D. and Foundation. ’ This paper is dedicated ven J. McNall, a colleague friend. a To whom correspondence

by National Tnstitutes T.E.M. is a member of the of parasitic Diseases supCatherine T. MacArthur to the memory of Dr. Stcat Stanford and a valued should

be addressed, 553

phofructokinase (4). These results suggest that phosphorylation of the key regulatory enzyme in glycolysis, phosphofructokinase, might be a mechanism for its modulation (5). We recently purified phosphofructokinase from the liver fluke and demonstrated that the enzyme was phosphorylated by cyclic AMP-dependent protein kinase (6). Phosphorylation of the enzyme resulted in its activation (6, 7). The question remained whether modulation of phosphofructokinase by phosphorylation occurs in viva (in the intact organisms). In this paper we present evidence that incubation of intact liver flukes with serotonin does, in fact, increase the state of phosphorylation as well as activating phosphofructokinase. The results indicate that phosphorylation of this enzyme is a relevant mechanism for its modulation in viva and that the effect of serotonin on glycolysis in these parasites is, at least in part, mediated through the serotonin signaling system that activates adenylate cyclase and cyclic AMP-dependent protein kinase. 0003-9861/89$3.00 Copyright@> All

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554

KAMEMOTO. EXPERIMENTAL

LAN.

PROCEDURES

Materials. F. hepatica were obtained from bile ducts of infected cattle at a local slaughterhouse. The organisms were maintained overnight in the laboratory as previously described (3) and stored at -80°C. New Zealand white rabbits were purchased from Nitabell. Other materials were obtained from the following sources: ATP, aldolase, glycerophosphate dehydrogenase, and triosephosphate isomerase from Boehringer-Mannheim; catalytic subunit of cyclic AMP-dependent protein kinase (bovine heart), protein A-enriched Staphlococcus au.reus cell suspension, protein A-Sepharose CL-4B, serotonin, AMP, Fru-6P, Fru-2,6-Pz from Sigma; and [r-“‘P]ATP (7000 Ci/ mmol) from ICN Radiochemicals. Preparation of antisera. Pure fluke phosphofructokinase (6) (200 rg) was emulsified with 2 vol of complete Freund’s adjuvant and injected subcutaneously at five sites in the back of the rabbit. A booster shot, consisting of 100 pg of phosphofructokinase in incomplete Fruend’s adjuvant, was administered 3 weeks later. The rabbit was bled from the ear after a week, then at 3-week intervals. A maximum of 20 ml of blood was collected per bleed. The blood was allowed to clot overnight, then centrifuged. Antisera were stored at -20°C. Antibody titer was determined as follows: 10 ~1 of antisera was incubated with 0.1 ml of protein A-enriched S. CGUTBUScells (10% wet wt/vol) suspended in 50 mM Mops4 buffer, pH 7.5. Formation of antibodyS. aureus complex was complete after 1 h at 4°C. The complexes were washed with buffer supplemented with 1 mg/ml bovine serum albumin to remove nonspecific binding, then suspended in buffer (minus albumin) to a cell concentration of 10% (w/v). The suspension (O-25 ~1) was incubated with 50 ~1 of fluke phosphofructokinase (0.2 mg/ml) for 30 min at room temperature with periodic mixing. Following centrifugation, phosphofructokinase activity in the supernatant was assayed as a measure of the amount of enzyme that was not immunoprecipitated.

Immunoprecipitation of phosphofructokinase in parasite extracts. Following incubation of the parasites, the extracts of the parasites were prepared as follows: lo-12 flukes (1 g wet wt) were homogenized in 5 ml of homogenization buffer (50 mM Mops, pH 7.5, 0.2 mM EGTA, 50 mM NaF, 1 mM sodium pyrophosphate, and 10 mM sodium vanadate) and centrifuged at 28,000~ for 20 min. Five milliliters of supernatant, containing phosphofructokinase, was incubated with 500 ~1 of the antibody-S aureu.s complex, prepared as

4 Abbreviations aminoethyl ether) dodecyl sulfate, fonic acid.

used: EGTA, ethylene glycol bis(PN,N’-tetraacetic acid; SDS, sodium Mops,3[N-morpholinolpropanesul-

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MANSOUR

described above. Following incubation for 1 h at 4°C the sample was centrifuged to pellet the S. aureus cells. Under these conditions, phosphofructokinase activity from the soluble fluke extracts was isolated in the pellet. Incubation ofjlukes with serotonin. Liver flukes, F. hepaticn, were dissected from bile ducts of infected cattle at a local slaughterhouse. The flukes were maintained overnight in saline media in order for the organisms to expel their gut content (2). Ten to twenty flukes were then placed in 200 ml of fresh media. Serotonin (1 mM, unless indicated otherwise) was added to the media. Following incubation for 15 min at 37°C the flukes were removed from the media, quick frozen with dry ice, and stored at -80°C for later analysis. Phosph,ofructokinase activity assay. Phosphofructokinase was assayed by coupling the enzyme with adolase, triosephosphate isomerase, and a-glycerophosphate dehydrogenase using the standard spectrophotometric method under assay conditions previously described (6). Where indicated, maximal enzyme activity was measured in a reaction mixture that contained 10 mM Fru-6-P, 1 mM ATP, 1 mM AMP, and 10 mM (NH&SO, at pH 8. Stoichiometry ofphosphorylution Phosphate incorporation into phosphofructokinase after separation on SDS-polyacrylamide gel electrophoresis was carried out as we described before (6), with minor modifications. Samples were centrifuged to remove the S. aureus cells prior to application onto the gel. The amount of phosphofructokinase in the gels was determined by protein staining and compared with known amounts of purified fluke phosphofructokinase which served as protein standard.

State of phosphorylation of phosphofructokinase in jtuke extracts. The state of phosphorylation of phosphofructokinase was determined by the procedure of back phosphorylation that was described by Nestler and Greengard (8). Phosphofructokinase was immunoprecipitated by the antiserum as described above. Phosphorylation of the immunoprecipitated enzyme was carried out by incubation with [y-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. The ATP concentration was 160 pM; otherwise, the conditions for phosphorylation of phosphofructokinase were the same as previously described (6). Following electrophoresis on an SDS polyacrylamide gel, phosphofructokinase (M, 83,000) was visualized by protein staining, and incorporation of 32P into the protein was detected by autoradiography. The protein band corresponding to phosphofructokinase was sliced from the gel and phosphorylation was quantitated by liquid scintillation counting. RESULTS

Nature of immunoprecipitated phosphcfructokinase from liverjluke extracts. Puri-

PHOSPHOFRUCTOKINASE

ACTIVITY

fication of phosphofructokinase from batches of parasites following incubation with serotonin would have required a large amount of material (30-60 g wet wt per each condition). Because of the difficulty of getting this amount of parasite material to carry out routine enzyme purification we devised an immunoprecipitation procedure to isolate and concentrate phosphofructokinase from liver fluke extracts. This procedure required significantly less parasite material. The procedure involved the use of polyclonal antibodies raised against purified fluke phosphofructokinase (6). The antibodies were absorbed onto protein A-enriched S. aureus cells as described under Experimental Procedures. Protein A binds immunoglobulin without affecting binding of antigen (9). The cells pellet and resuspend easily, thus allowing for the manipulation of small quantities of immunoconjugates as described above. The key finding that formed the basis for the utility of this isolation scheme was that formation of the phosphofructokinase-antibody-protein A-S. aureus complex had only a slight inhibitory effect on phosphofructokinase activity. Following resuspension, the immunoprecipitated enzyme was assayed for activity by the standard spectrophotometric method. Mean recovery of phosphofructokinase activity in the immunoprecipitated pellet was 73%, with a range of 6684% (14 samples). S. aureus alone had no phosphofructokinase activity under these experimental conditions. Furthermore, the S. aureDs cells could be replaced by protein A-Sepharose CL-4B beads. The possibility of nonspecific binding of fluke phosphofructokinase to S. aureus or Sepharose was ruled out since preimmune sera did not immunoprecipitate any enzyme. The resuspended immunoprecipitated phosphofructokinase retained allosteric properties that are characteristic of fluke phosphofructokinase (7). Fluke phosphofructokinase is distinct from the mammalian enzyme in that inhibition by ATP is not limited to assays at acidic pH, but can also occur under alkaline conditions. Similarly, immunoprecipitated enzyme was inhibited by ATP at alkaline pH (8.0). The

IN

F~~scioh

555

kpafiw

PFK-

A

6

C

0

FIG. 1. SDS-polyacrylamide gel electrophoresis and autoradiography. Samples were electrophoresed on 7.5% polyacrylamide gels and stained with Coomassie blue (lanes A-C). (A) Protein standards and their molecular weights, from top to bottom: fi-gaiactosidase (116,250), phosphorylase h (97,400), liver fluke phosphofructokinase, PFK (83,000), and hovine serum albumin (66,200); (B) immunoprecipitated phosphofructokinase; (C) immunoprecipitated phosphofructokinase incubated with catalytic subunit of cyclic AMP-dependent protein kinase and [y“2P]ATP: (D) autoradiogram of gel lane shown in C.

ATP concentration required for inhibition of the enzyme to half of its maximal velocity was 1.3 mM, a value that is similar to 1.1 mM obtained for pure phosphofructokinase (Fig. 8 in Ref. (7)). Two potent activators of fluke phosphofructokinase, AMP and Fru-2,6-P,, also had the same effects on the immunoprecipitated enzyme. At saturating concentrations of either AMP (1 mM) or Fru-2,6-Pz (0.1 mM), both pure fluke phosphofructokinase and immunoprecipitated enzyme were no longer inhibited by ATP. Immunoprecipitated phosphofructokinase was a substrate for phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. The resuspended immunoprecipitated enzyme was incubated with [y-“ZP]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. Figure 1 illustrates a representative experiment for SDS-polyacrylamide gel electrophoresis and autoradiography of the immunoprecipitated phosphofructokinase from fluke extracts. Bands for the enzyme without phosphorylation (lane El) and after phosphorylation (lane C) show that the phosphorylated enzyme had a molecular weight identical to that of the stan-

556

KAMEMOTO,

LAN,

TIME (min)

FIG. 2. Time course of phosphorylation. Phosphofructokinase from control and serotonin-treated batches of flukes were immunoprecipitated as described above. The immunoprecipitated enzyme was subjected to the procedure of back phosphorylation in the presence of [T-~P]ATP and catalytic subunit of cyclic AMP-dependent protein kinase. At the indicated times, the reaction was stopped with 10 mM EDTA, and the level of phosphorylation was measured as described under Experimental Procedures. Graphs for levels of phosphorylation of phosphofructokinase in control (a) and following incubation with serotonin (A) are shown.

dard enzyme (lane A). The autoradiogram of the phosphorylated enzyme is shown in lane D. The protein band near the bottom of the gel is that of immunoglobulin. Immunoprecipitation was specific for phosphofructokinase with the exception of a minor contaminant (Mr 102,000) that was also phosphorylated (Fig. 1). The identity of this band is unknown. However, its phosphorylation is not regulated by serotonin (data not shown), unlike phosphorylation of phosphofructokinase (as dephosphate scribed below). Maximum incorporation into immunoprecipitated phosphofructokinase ranged from 0.2 to 0.3 mol P/mol of protomer. This is shown in the results summarized in Fig. 2 (control curve). This low stoichiometry is characteristic of pure fluke phosphofructokinase and has been fully examined before (7). Phosphcwylation

of phosphofructokinase

in viva. Preliminary studies demonstrated that uptake of inorganic 32P from culture 5 T. E. Mansour,

unpublished

observations.

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MANSOUR

media by whole flukes was inefficient.5 This compromised our attempts to label phosphofructokinase and to measure the level of phosphorylation of the enzyme in vivo. Consequently, the method of “back phosphorylation” was used to measure the level of enzyme phosphorylation (8). It provided greater sensitivity than labeling with inorganic 32P. Whole flukes were incubated in media. Serotonin was added to the incubation media as a potential activator of phosphofruetokinase phosphorylation. Following incubation, phosphofructokinase was immunoprecipitated, isolated, and then back phosphorylated, by incubation with [T-~P]ATP and protein kinase as described above. A decrease in the level of back phosphorylation following incubation with serotonin was interpreted as an in ,vivo increase in the level of phosphorylation of the enzyme. The sensitivity of this procedure is high since there are no permeability barriers limiting access of radiolabel or endogenous pools to decrease the specific radioactivity of [T-~~P]ATP. The results summarized in Fig. 2 show the time course of phosphorylation of the immunoprecipitated phosphofructokinase. Maximum phosphate incorporation in serotonintreated enzyme samples was only 75% of control samples. Given the results of five separate experiments, phosphate incorporation in phosphofructokinase from serotonin-treated flukes, relative to control enzyme, was 74 ? 7.7% (standard deviation). That means that the level of phosphorylation as a result

of serotonin

action in vivo

was increased to the extent of 25% of maximal phosphorylation. Since this value was determined to be 0.2 to 0.3 mol P/mol of protomer (see above), the amount of incorporated phosphate under in vivo conditions is 0.05 to 0.075 mol P/m01 enzyme protomer. A conclusion can be drawn that incubation of intact flukes with serotonin increases the level of phosphorylation of phosphofructokinase. Efect of serotonin and of back phosphorylation on immunoprecipitated phosphofructokinase activity. Samples of control and

serotonin-treated immunoprecipitated enzyme were assayed under conditions optimal for maximal activity and those opti-

PHOSPHOFRUCTOKINASE TABLE

ACTIVITY

I

EFFECT OF SEROTONIN ON PHOSPHOFRUCTOKINASE ACTIVITY IN INTACT LIVER FLUKES

Incubation conditions of liver flukes Control

Back phosphorylation -

+ 1 mM Serotonin

-

+

Phosphofructokinase activity (S VnlJ Fru-6-P (mM) 10

15

20

6 36 20 43

14 51 29 50

26 60 34 56

Note. Liver flukes were incubated with or without serotonin. Phosphofructokinase was assayed in immunopreeipitated enzyme before and after phosphorylation with the catalytic subunit of cyclic AMP-dependent protein kinase and ATP. Methods for incubation of intact parasites, immunoprecipitation of the enzyme, and phosphorylation were as described under Experimental Procedures. Maximal enzyme activity (100%) was determined in the presence of AMP and (NH,)2S04 as described under Experimental Procedures.

ma1 for allosteric kinetics. Maximal activity was unaffected following incubation with serotonin (data not shown). However, under conditions ideal for detecting allosteric kinetics (i.e., nonsaturating fructose &phosphate and inhibitory ATP concentrations), phosphofructokinase from serotonin-treated liver flukes was more active than enzyme from control parasites. A representative example of five experiments is shown in Table I. The effect of incubation of the flukes with serotonin was greater than threefold at 10 mM fructose 6phosphate, and was twofold at 15 mM of the substrate. The stimulatory effect of serotonin was less pronounced at 20 mM fructose 6-phosphate, the highest substrate concentration used. These results are consistent with the conversion from sigmoidal toward hyperbolic kinetics as a result of phosphorylation (see Fig. 2, Ref. (7)). In addition, phosphofructokinase from serotonin-treated flukes was less sensitive to inhibition by ATP than was the enzyme from control parasites (data not shown).

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Fnsciola hepaticn

557

Back phosphorylation of immunoprecipitated phosphofructokinase increased the activity of the enzyme from control parasites several-fold. It also activated the enzyme from serotonin-stimulated parasites, but to a lesser degree (Table I). This difference in the effect of back phosphorylation on enzyme activity in the two samples reflects the fact that the extent of back phosphorylation of phosphofructokinase in serotonin-treated flukes was less than that in the control group. The end result was that there was no significant difference in activity between control and serotonin-treated flukes following back phosphorylation. The effect of different concentrations of serotonin on both activity and phosphorylation level of phosphofructokinase in intact organisms was examined. These results are summarized in Fig. 3. An increase in phosphofructokinase activity and a decrease in back phosphorylation level, designating an in vivo increase in enzyme phosphorylation, was observed at each of the three serotonin concentrations tried. The magnitude of these effects increased as a function of serotonin concentration, and was maximal at 1 mM of the indolamine.

3o II”,’

[Serotom]

(mM)

FIG. 3. Levels of phosphorylation and activity of phosphofructokinase at different serotonin concentrations. Levels of phosphorylation and enzyme activity were determined in immunoprecipitated phosphofructokinase from batches of flukes incubated at different serotonin concentrations. Graphs for phosphofructokinase activity (A) and levels of phosphorylation (A) are shown.

558

KAMEMOTO,

LAN,

DISCUSSION

Phosphofructokinase is known to be a key enzyme in the regulation of glycolysis in both aerobic and anaerobic organisms. The enzyme appears to play a special role in the regulation of glycolysis in anaerobic organisms such as the liver fluke F. hepatica. Serotonin, an epinephrine-like hormone in this organism, was shown to stimulate glycolysis by activating phosphofructokinase. The enzyme in the liver fluke is endowed with two mechanisms for its modulation, regulation by allosteric modifiers and regulation by phosphorylation with cyclic AMP-dependent protein kinase (6,7). We previously demonstrated a correlation between phosphate incorporation and phosphofructokinase activation in the pure enzyme system. The significance of phosphorylation as an in vtuo mechanism for regulation of phosphofructokinase is corroborated by our present investigation on intact liver flukes. Phosphofructokinase from serotonin-treated flukes did have higher activity. Activation of the enzyme was shown to parallel an increase in its phosphorylation level. Evidence for the latter effect was obtained through the use of the technique of back phosphorylation (8). Phosphorylation appears to be at a site that is recognized by the cyclic AMP-dependent protein kinase. It is interesting to note that an increase of 0.08 mol of phosphate per protomer following serotonin treatment of intact organisms (Fig. 2) resulted in a threefold increase in enzyme activity (Table I). I72 GVO changes in phosphofructokinase activity can therefore occur as a result of minor changes in the level of phosphorylation of the enzyme. Our previous studies on the pure enzyme (6) showed that maximal phosphorylation by cyclic AMP-dependent protein kinase resulted in the addition of only 0.22 mol of phosphate per enzyme protomer. This low level of phosphorylation resulted in a marked activation of the enzyme. Results from in GVO experiments described in this report, as well as previous studies with the pure enzyme, point toward the conclusion that phosphorylation is an important modulator of the liver

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MANSOUR

fluke phosphofructokinase. A corresponding role for phosphorylation in the regulation of mammalian phosphofructokinase has yet to be demonstrated (10-13). In contrast, phosphorylation of phosphofructokinase isolated from the parasitic nematode, Ascaris suum, resulted in an increase in enzyme activity (14,15). It still remains to be seen whether this regulatory mechanism in the case of Ascaris enzyme is linked to a hormonal receptor such as serotonin receptors in F. hepatica. There is a similarity between the two parasites in that both are strict anaerobes. Their complete dependence on anaerobic carbohydrate metabolism for energy may be related to the need for a greater control over phosphofructokinase by covalent as well as noncovalent mechanisms. Protein phosphorylation is recognized as a common mechanism by which hormones and other extracellular effecters can regulate intracellular metabolism. As stated earlier, serotonin has hormone-like effects on the carbohydrate metabolism of the liver fluke. Previous results from our laboratory on intact organisms showed that serotonin increases the levels of cyclic AMP (16) and activates protein kinase (17). The above results indicate that the effect of serotonin on the fluke phosphofructokinase is mediated at least partially by phosphorylation through the activity of cyclic AMP-dependent protein kinase. These results are analogous to epinephrine-induced phosphorylation of glycogen phosphorylase kinase (18,19), a classical model for the hormonal regulation of an enzyme. ACKNOWLEDGMENT We thank Dr. Howard reading of the manuscript.

Schulman

for

his critical

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PHOSPHOFRUCTOKINASE 5. MANSOUR, T. E., AND MANSOUR, J. M. (1962) X. Biol. Ch.enz. 237,629-634. 6. KAMEMOTO, E. S., AND MANSOUK. T. E. (1986) J. Biol. Chern. 261,4346-4351. 7. KAMEMOTO, E. S., ILTZSCH, M. H., LAN, L., AND MANSOUR, T. E. (1987) A&. Biochem. Biophys. 258,101-111. 8. NESTLER, E. J., AND GREENGARD, P. (1982) J. Neurosci. 2,1011-1023. 9. KESSLER, S. W. (1975) J. Zmmunol. 115,1617-1624. 10. PILKIS, S. J., EL-MAGHRABI, M. R., PILKIY, J., AND CLAUS, T. H. (1982) Arch. Biochem. Biophys. 215,379-389. 11. FOE, L. G., AND KEMP, R. G. (1982) J. Biol. Chm. 257,6368-6372. 12. SAKAKIBARA, R., AND UYEDA, K. (1983) J. Biol. Chertc. 258,8656-8662.

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