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Protein phosphatases type 1 and type 2 in Drosophila melanogaster
Comp. Biochem. PhysioL Vol. 87B, No. 4, pp. 85%861, 1987 Printed in Great Britain
0305-0491/87 $3.00+0.00 (c~ 1987 PergamonJournals Ltd
P R O T E I N P H O S P H A T A S E S TYPE 1 A N D TYPE 2 IN DROSOPHILA MELANOGASTER VIKTOR DOMBR.~.DI*, PETER FRIEDRICHt and GEORG BOT Institute of Medical Chemistry, University School of Medicine, Bern t6r 18/B, Debrecen, H-4026, Hungary; and tInstitute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, P.O. Box 7, H-1502, Hungary (Tel: 361-668-858)
(Received 8 September 1986) Abstract--1. The heat-stable protein inhibitors (inhibitor-I and -2) isolated from rabbit skeletal muscle proved to be suitable tools for the classification of protein phosphatases in Drosophila melanogaster. 2. Inhibitor-sensitive type 1 phosphatase represented the great majority of phosphorylase phosphatase activity during all stages of fruit fly development, inhibitor-insensitive type 2 phosphatase was presented in a latent form. 3. Only the nervous tissue of the flies contained substantial amount of spontaneously active type 2 phosphorylase phosphatase. 4. (NH4)2SO4 and ethanol treatment converted large molecular mass phosphatases to an Mr ~ 37,000 enzyme form that was resolved into type 1 and 2 catalytic subunits by heparin-Sepharose chromatography.
mammalian inhibitors may not affect the shrimp enzymes' (Thoen et al., 1985). To our knowledge the presence of inhibitor-sensitive type 1 phosphatase in any invertebrate source has not yet been proven. In the present communication we describe that rabbit skeletal muscle inhibitors-1 and -2 can be used for the classification of protein phosphatases in insect samples and demonstrate the presence of types 1 and 2 phosphatases in Drosophila melanogaster.
INTRODUCTION Regulation of enzyme activity by reversible phosphorylation-dephosphorylation is a universal phenomenon in all eukaryotic cells (Cohen, 1985). It has been shown that nearly all dephosphorylation reactions taking place in mammalian tissues could be explained by the action of only four protein phosphatases (Ingebritsen and Cohen, 1983). These were conveniently divided into two classes. Type 1 phosphatases are inhibited by the thermostable proteins inhibitor-1 and inhibitor-2, furthermore, they specifically dephosphorylate the ~ subunit of rabbit skeletal muscle phosphorylase kinase. In contrast, type 2 phosphatases are not affected by the heatstable inhibitors and preferentially dephosphorylate the ~ subunit of phosphorylase kinase (Ingebritsen and Cohen, 1983). The presence of phosphorylase phosphatase (EC 3.1.3.17) in an insect was directly proven by Ashida and Wyatt (1979). Recently, a phosphorylase phosphatase has been partially purified from the fat body of Phylosamia cyntha by Hayakawa (1985). On the basis of its ATP sensitivity it was suggested that this preparation contained type 2 phosphatase activity (Hayakawa, 1985), however, the criteria introduced by Ingebritsen and Cohen (1983) were not tested. In another recent publication (Thoen et al., 1985) five phosphorylase phosphatases have been separated by DEAE-cellulose chromatography from the gastrulae of the crustacean species Artemia salina. Two of these phosphatases were characterized and were found to be inhibitor-insensitive type 2 enzymes. In this work the possibility remained open that 'the lack of effect of inhibitor-1 and -2 might be due to the fact that the
MATERIALSAND METHODS
Materials Heparin-Sepharose CL-6B and Sephacryl S-200 superfine from Pharmacia (Uppsala) were employed. The following molecular mass markers were obtained from Serva (Heidelberg): myoglobin (Mr~ 18,000); carbonic anhydrase (Mr ~ 29,000); ovalbumin (Mr ~ 45,000); bovine serum albumin (Mr ~ 68,000); aldolase (M~ ~ 147,000) and catalase (Mr ~ 240,000). [y-32P]ATPwas the product of the Institute of Isotopes, Hungarian Academy of Sciences (Budapest). Glycogen was prepared from rabbit liver according to Mordoch et al. (1966). Other chemicals were purchased from Reanal (Budapest). Phosphorylase b was isolated from rabbit skeletal muscle (Fischer and Krebs, 1962) and was converted to phosphorylase a with rabbit muscle phosphorylase kinase and ATP or [y-32p]ATP (Krebs and Fischer, 1962). The specific radioactivity of 32p labeled phosphorylase a was 3 × 106Bq/mgprotein. Phosphorylated inhibitor-1 and nonphosphorylated inhibitor-2 were purified from rabbit skeletal muscle as described by Shenolikar and Ingebritsen (1984).
*To whom correspondence should be addressed. Abbreviations: Mops, 3-(N-morpholino)propanesulfonic acid; Tris, tris-(hydroxymethyl)-aminomethane;EDTA, ethylenediamine-tetraacetic acid. C.B.P. S7/4~-N
857
Animals and sample preparation Drosophila melanogaster (Canton-S strain) was reared on standard cornmeal medium at 25°C according to Ashburner and Thompson (1978). Eggs (~ 3 hr old) and first instar larvae (1 day old) were rinsed off the surface of the medium by a Drosophila ringer, containing 5 mM Mops, pH 6.8, 10 mM D-glucose, 128 mM NaC1 and 4.7 mM KC1, and were washed three times in the same solution. Third instar larvae (84 days old) and pupae
858
VIKTOR DOMBR~,DI et al. 100
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100
800
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100,
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substrate and was carried out at 30°C. 20 #1 samples were withdrawn at appropriate time intervals (2-10 min), diluted 20-fold in a solution containing 40 mM ~-glycerophosphate, pH6.8, 2mM EDTA, 10mM 2-mercaptoethanol and 100 mM NaF, and the residual phosphorylase a activity was determined (Illingworth and Cori, 1953). The dephosphorylation reactions were linear up to 20% conversion of the substrate, accordingly, the homogenates were diluted with the homogenisation buffer so that no more than 20% of phosphorylase a was dephosphorylated within 10min. One unit of phosphorylase phosphatase liberates 1 nmol phosphate from the substrate within 1 min under these experimental conditions. The validity of phosphatase assay was proven by two facts: (i) 20 mM NaF, a well known inhibitor of phosphorylase phosphatase, was sufficient to block the interconversion of phosphorylase a (cf. Thoen et al., 1985). (ii) More than 95% of the radioactivity liberated from a2p labeled phosphorylase a was extracted as phosphomolybdate by organic solvent, indicating that inorganic phosphate and not phosphopeptide(s) was produced in the assay (Shenolikar and Ingebritsen, 1984). Inhibitors-I and -2 were assayed with the catalytic subunit of phosphatase-1 isolated from rabbit skeletal muscle as described by Shenolikar and Ingebritsen (1984). One unit of
2O
Vo ~ . ~
1
10 INHIBITOR-2
100
400
~= ~
~
0.6
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Fig. 1. Effect of rabbit muscle inhibitors-I and -2 on phosphorylase phosphatase activity of crude D. melanogaster imago extracts. The phosphatase activity of 10-fold diluted extracts was determined after preincubation in the presence or absence of the heat-stable inhibitor proteins. The activity without addition (0.4-0.6 U/ml) was taken as 100%. Each point represents the mean of three independent determinations; the standard deviation was less than + 6%.
04
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(1 day after pupation) were collected from the wall of the culture jar. Adult flies (2 weeks old) were harvested after quick anaesthesia with diethyletber. The brain of third instar larvae was dissected under a stereomicroscope. The neck of imagos was broken by mechanical agitation in liquid nitrogen and the heads were separated from other body parts by sequential seiving. All the above samples were stored in liquid nitrogen until homogenization. The samples were homogenized in 40 mM Tris buffer adjusted to pH 7.2 with 0.1 M HCI at room temperature and containing 2 mM EDTA, 10 mM 2-mercaptoethanol, 5 mM benzamidine, 1 mM o-phenantroline and 0.1 mM phenylmethylsulfonylfluoride by a motor-driven Teflon-glass Potter device with 25 strokes at 0°C. The homogenates were centrifuged at 10,000g (r~. 6cm) and 0°C for 10 min and supernatants were filtered through glass wool. Assays Protein phosphate activity was measured with rabbit skeletal muscle phosphorylase a that was shown to be a good substrate of insect phosphatases (Ashida and Wyatt, 1979; Hayakawa, 1985). The assay was based on the procedure of Brandt et al. (1975). The reaction mixture was composed of 100/~1 homogenate, 50 #1 40 mM Tris x HC1, pH 7.2, 2 mM EDTA, 10 mM 2-mercaptoethanol containing various additions and 100#1 25 U/ml rabbit muscle phosphorylase a and 12.5 mM caffeine dissolved in the same buffer. The reaction was started by the addition of the
1.0
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Fig. 2. Gel filtration of fruit-fly protein phosphatases. A 1.6 x 33 cm Sephacryl S-200 column was equilibrated and developed with 50 mM imidazole, pH 7.2, buffer containing 5 mM EDTA, 10 mM 2-mercaptoethanol, 0.1 M NaC1 and 10% glycerol at 6 _+2°C. The column was run at 10ml/hr flow rate and fractions of 1 ml were collected. 0.5 ml crude imago extract containing 8.2 mg protein (upper panel) or 0.5ml (NH4)2SO4, ethanol-treated extract containing 0.25 mg protein (lower panel) was applied onto the column and the phosphorylase phosphatase activity of the fractions was determined. Arrows indicate the elution position of marker proteins (molecular mass in kilodalton).
Protein phosphatases in an insect the inhibitors causes 50% inhibition of 20 mU phosphatase in the above assay system, The effect of inhibitors on Drosophila phosphatases was studied after 10min preincubation at 30°C with the diluted homogenates. Protein was measured by the dye-binding method of Bradford (1976). Bovine serum albumin obtained from Calbioehem (Los Angeles, CA) was used as standard and its concentration was checked spectrophotometrically (A~ = 6.5).
859
phosphatase (Ingebritsen et aL, 1980; Silberman et al., 1984) we assumed that the low molecular mass
phosphatase was composed of two different types of catalytic subunit and that the subunits could be separated by heparin-Sepharose chromatography (G-ergely et al., 1984). Indeed, two peaks of phosphatase activity were obtained after heparinSepharose chromatography (Fig. 3). The portion of phosphatase that did not bind to the affinity matrix was ..ot influenced by inhibitor-l, while the portion RESULTS bound to the column and eluted at 0.19 M NaCI was Inhibitor-sensitive and -insensitive protein phos- fully inactivated by the inhibitor protein. phatases The combination of (NH4)2SO4 and ethanol preInhibitors-1 and -2 isolated from rabbit skeletal cipitation with heparin-Sepharose chromatography muscle inactivated phosphorylase phosphatase in yielded a partially purified type 1 catalytic subunit crude extracts of adult Drosophila (Fig. 1). The preparation free of type 2 phosphatase. An 800--1000inhibition was concentration dependent; 800 U/ml fold purification was achieved by this simple proinhibitor- 1 or 400 U/ml inhibitor-2 caused practically cedure. total inactivation of the enzyme. The presence of inhibitor-insensitive phosphatase in the same extract was revealed by a so called Developmental study All of the above experiments were carried out with 'activating' treatment. Precipitation of the homogenate (6-8 mg/ml protein) by (NH4)2SO4 and ethanol samples obtained from the whole body of adult according to Brandt et al. (1975) enhanced phos- insects. The changes in phosphorylase phosphatase phorylase phosphatase activity to 127 _ 11% (N = 5) activity during the development of the flies was also of the original value. After activation only 60-70% of studied (Table 1). The specific activity of soluble the enzyme activity was inhibited by 1000U/ml phosphatase was the highest in early developmental inhibitor-l. The (NH4)2SO4 and ethanol treatment stages, i.e. in eggs and first instar larvae and was the converted larger molecular mass forms of phos- lowest in pupae. In all developmental stages the great majority of spontaneously active enzyme was sensiphatase into a form of Mr ~ 37,000 (Fig. 2.). tive to inhibitor-1. Two samples enriched in nervous Separation of two catalytic subunits tissue were also analysed. The larval brain and imago Based on analogies with the mammalian protein head contained higher specific activity of phosphatase
1-25 ~-
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10
20 FRACTION
30
40
1
lI
°
50
NUMBER
Fig. 3. Affinity chromatography of D. melanogaster protein phosphatase catalytic subunits. A I x 29 cm heparin-Sepharose column was equilibrated with 50 mM imidazole, pH 7.2, buffer containing 5 mM EDTA and 10mM 2-mercaptoethanol at 6+2°C with 20ml/hr flow rate. 50ml of (NH4)2SO4, ethanol-treated fruit fly extract (0.35 mg/ml protein) dialysed against the column buffer was loaded on the column. The column was washed with the same buffer and eluted with a linear 0-0.5 M NaC1 gradient. The volume of fractions 1-18 was 5.1 ml and of fractions 19-52 was 1.6 ml. The fractions were assayed for phosphorylase phosphatase activity in the absence (O) and in the presence ( 0 ) of 1000U/ml inhibitor-l, and for protein ( x ). The figure is representative of five similar separations.
860
VIKTOR DOMBRId)I et al.
Table 1. Levels of protein phospohatases at various developmental stages of D. melanogaster Phosphorylase phosphatase activity Stage Total (U/mg protein) Type 1 (%) Egg 1.4 + 0.3 84 ± 8 First instar larva 1.4 ± 0.2 99 ± 4 Third instar larva 0.75 + 0.08 90 ± 5 Puparium 0.27 ± 0.05 83 ± 7 Imago 0.76 ± 0.17 96 ± 4 Brain of third 2.3 ± 0.7 60 ± 13 instar larva Head of imago 1.6 ± 0.5 70 ± 13 Means of 7-11 determinations + standard deviations are given. Whole bodies were used unless otherwise indicated. The specific activity of phosphorylase phosphatase was measured in crude extracts. The percentage of type 1 phosphatase corresponds to the activity inhibited by 800 U/ml heat stable inhibitor-1.
than the respective whole bodies and 30-40% of this activity was not affected by inhibitor-1 (Table 1). DISCUSSION
Rabbit muscle inhibitors-1 and -2 caused a dosedependent inactivation of Drosophila phosphorylase phosphatase. The inhibition proved to be selective. The catalytic subunit that was bound to heparinSepharose was totally inhibited while the unbound catalytic subunit was not influenced. It is known that heparin-Sepharose specifically adsorbs the catalytic subunit of type 1 phosphatase and does not interact with type 2 phosphatases (Gergely et al., 1984). The findings indicate that the classification of protein phosphatases originally designed for mammalian tissues (Ingebritsen and Cohen, 1983) can be extended to the realm of insects, and that the thermostable inhibitor proteins isolated from rabbit skeletal muscle can be widely used. Our results substantiate the classification of the two inhibitor-insensitive shrimp phosphatase as type 2 enzymes (Thoen et al., 1985) and will facilitate the detection of type 1 phosphatases in non-mammalian tissues. The fact that rabbit inhibitor proteins specifically interact with type 1 fruitfly phosphatase both in crude extracts and in a partially purified catalytic subunit preparation suggests that the regulation of type 1 phosphatase by the inhibitors must have been well conserved during evolution. Another similarity between the well-known mammalian and the here characterized insect phosphatases is the activation and dissociation of larger molecular mass enzyme forms into Mr ~ 37,000 catalytic subunits by (NH4)2SOq-ethanol treatment (Brandt et al., 1975). We found significant alterations in phosphorylase phosphatase activity during the development of Drosophila, however, in all stages type 1 phosphatase was predominant. Only in brain and head samples were considerable amounts of spontaneously active type 2 phosphatase present. These results are in agreement with the findings that many mammalian tissues contain type 2 phosphatase in a latent form (Di Salvo et al., 1984; Schlender and MeUgren, 1984; Erd6di et al., 1985) and that rabbit brain is a rich source of active type 2 p h o s p h o r y l a s e p h o s p h a t a s e (Ingebritsen et al., 1983). The c o o r d i n a t e d control o f type 1 p h o s p h a t a s e by inhibitor-1 is well d o c u m e n t e d (cf. Cohen, 1985). The p r e d o m i n a n c e of type 1 p h o s p h a t a s e in Droso-
phila makes it probable that the same mechanism can operate also in this insect. Acknowledgements--Thanks are due to Dr Ferenc Erd6di, Institute of Medical Chemistry, University School of Medicine, Debrecen, for the preparation of inhibitor-I and -2. The excellent technical assistance of Mrs. Margit Bir6 and Mrs. Agnes Csurg6 is acknowledged. This work was supported in part by the Hungarian Ministry of Health. Note added in proof. After submission of this manuscript we became aware of a work by S. Orgad, Y. Dudai and P. Cohen (Eur. J. Biochem., in press) in which these authors also detected both type 1 and type 2 protein phosphatases in Drosophila heads using heat-stable inhibitor-I and -2 from rabbit muscle. We thank Dr Philip Cohen for kindly sending us the manuscript before publication.
REFERENCES
Ashburner M. and Thompson Jr. J. N. (1978) The laboratory culture of Dorosphila. In The Genetics and Biology of Drosophila (Edited by Ashburner M. and Wright T. R. F.), Vol. 2, pp. 2-109. Academic Press, New York. Ashida M. and Wyatt O. R. (1979) Properties and activation of phosphorylase kinase from silkmoth fat body. Insect Biochem. 9, 403-409. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-253. Brandt H., Capulong Z. L. and Lee E. Y. C. (1975) Purification and properties of rabbit liver phosphorylase phosphatase. J. biol. Chem. 250, 8038-8044. Cohen P. (1985) The role of protein phosphorylation in the hormonal control of enzyme activity. Eur. J. Biochem. 151, 439-448. Di Salvo J., Gifford D. and Kokkinakis A. (1984) Modulation of aortic protein phosphatase activity by polylysine. Proc. Soc. exp. Biol. Med. 177, 24-32. Erdfdi F., Csortos Cs., Bot G. and Gergely P. (1985) Separation of rabbit liver latent and spontaneously active phosphorylase phosphatases by chromatography on heparin-Sepharose. Biochem, biophys. Res. Commun. 128, 705-712. Fischer E. H. and Krebs E. G. (1962) Muscle phosphorylase b. Meth. Enzymol. 5, 369-373. Gergely P., Erd6di F. and Bot G. (1984) Heparin inhibits the activity of protein phosphatase-1. FEBS Lett. 169, 45-48. Hayakawa Y. (1985) Activation mechanism of insect fat body phosphorylase by cold. Phosphorylase kinase, phosphatase and ATP level. Insect Biochem. 15, 123-128. lllingworth B. and Cori G. T. (1953) Crystalline muscle phosphorylase b. In Biochemical Preparations (Edited by Snell E. E.), Vol. 3, pp. 1-9. John Wiley, New York. Ingebritsen T. S. and Cohen P. (1983) Protein phosphatases: Properties and role in cellular regulation. Science 221, 331-338. Ingebritsen T. S., Foulkes J. G. and Cohen P. (1980) The broad specificity protein phosphatase from mammalian liver. Separation of the Mr ~ 35,000 catalytic subunit into two distinct enzymes. FEBS Lett. 119, 9-15. Ingebritsen T. S., Stewart A. A. and Cohen P. (1983) The protein phosphatases involved in cellular regulations6. Measurement of type-I and type-2 protein phosphatases in extracts of mammalian tissues; an assessment of their physiological roles. Eur. J. Biochem. 132, 297-307. Krebs E. G. and Fischer E. H. (1962) Phosphorylase kinase from rabbit skeletal muscle. Meth. Enzymol. 5, 373-376. Mordoch J., Krisman C. R. and Leloir U F. (1966) Further
Protein phosphatases in an insect studies on high molecular weight glycogen. Arch. Biochem. Biophys. 113, 265-272. Schlender K. K. and Mellgren R. L. (1984) Isolation of historic HI-stimulated phosphoprotein phosphatase from kidney and skeletal muscle. Proc. Soc. exp. Biol. Med. 177, 17-23. Shenolikar S. and Ingebritsen T. S. (1984) Protein (serine and threonine) phosphate phosphatases. Meth. Enzymol. 107, 102-129.
861
Silberman S. R., Speth M., Nemani R., Ganapathi M. K., Dombrfidi V., Paris H. and Lee E. Y. C. (1984) Isolation and characterization of rabbit skeletal muscle protein phosphatases C-I and C-II. J. biol. Chem. 259, 2913-2922. Thoen C., Van Hove, Cohen P. and Slegers H. (1985) Identification of protein phosphatases dephosphorylating mRNP proteins from cryptobiotic gastruale of the brine shrimp A. salina. Biochem. biophys. Res. Commun. 131, 84-90.
0305-0491/87 $3.00+0.00 (c~ 1987 PergamonJournals Ltd
P R O T E I N P H O S P H A T A S E S TYPE 1 A N D TYPE 2 IN DROSOPHILA MELANOGASTER VIKTOR DOMBR.~.DI*, PETER FRIEDRICHt and GEORG BOT Institute of Medical Chemistry, University School of Medicine, Bern t6r 18/B, Debrecen, H-4026, Hungary; and tInstitute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, P.O. Box 7, H-1502, Hungary (Tel: 361-668-858)
(Received 8 September 1986) Abstract--1. The heat-stable protein inhibitors (inhibitor-I and -2) isolated from rabbit skeletal muscle proved to be suitable tools for the classification of protein phosphatases in Drosophila melanogaster. 2. Inhibitor-sensitive type 1 phosphatase represented the great majority of phosphorylase phosphatase activity during all stages of fruit fly development, inhibitor-insensitive type 2 phosphatase was presented in a latent form. 3. Only the nervous tissue of the flies contained substantial amount of spontaneously active type 2 phosphorylase phosphatase. 4. (NH4)2SO4 and ethanol treatment converted large molecular mass phosphatases to an Mr ~ 37,000 enzyme form that was resolved into type 1 and 2 catalytic subunits by heparin-Sepharose chromatography.
mammalian inhibitors may not affect the shrimp enzymes' (Thoen et al., 1985). To our knowledge the presence of inhibitor-sensitive type 1 phosphatase in any invertebrate source has not yet been proven. In the present communication we describe that rabbit skeletal muscle inhibitors-1 and -2 can be used for the classification of protein phosphatases in insect samples and demonstrate the presence of types 1 and 2 phosphatases in Drosophila melanogaster.
INTRODUCTION Regulation of enzyme activity by reversible phosphorylation-dephosphorylation is a universal phenomenon in all eukaryotic cells (Cohen, 1985). It has been shown that nearly all dephosphorylation reactions taking place in mammalian tissues could be explained by the action of only four protein phosphatases (Ingebritsen and Cohen, 1983). These were conveniently divided into two classes. Type 1 phosphatases are inhibited by the thermostable proteins inhibitor-1 and inhibitor-2, furthermore, they specifically dephosphorylate the ~ subunit of rabbit skeletal muscle phosphorylase kinase. In contrast, type 2 phosphatases are not affected by the heatstable inhibitors and preferentially dephosphorylate the ~ subunit of phosphorylase kinase (Ingebritsen and Cohen, 1983). The presence of phosphorylase phosphatase (EC 3.1.3.17) in an insect was directly proven by Ashida and Wyatt (1979). Recently, a phosphorylase phosphatase has been partially purified from the fat body of Phylosamia cyntha by Hayakawa (1985). On the basis of its ATP sensitivity it was suggested that this preparation contained type 2 phosphatase activity (Hayakawa, 1985), however, the criteria introduced by Ingebritsen and Cohen (1983) were not tested. In another recent publication (Thoen et al., 1985) five phosphorylase phosphatases have been separated by DEAE-cellulose chromatography from the gastrulae of the crustacean species Artemia salina. Two of these phosphatases were characterized and were found to be inhibitor-insensitive type 2 enzymes. In this work the possibility remained open that 'the lack of effect of inhibitor-1 and -2 might be due to the fact that the
MATERIALSAND METHODS
Materials Heparin-Sepharose CL-6B and Sephacryl S-200 superfine from Pharmacia (Uppsala) were employed. The following molecular mass markers were obtained from Serva (Heidelberg): myoglobin (Mr~ 18,000); carbonic anhydrase (Mr ~ 29,000); ovalbumin (Mr ~ 45,000); bovine serum albumin (Mr ~ 68,000); aldolase (M~ ~ 147,000) and catalase (Mr ~ 240,000). [y-32P]ATPwas the product of the Institute of Isotopes, Hungarian Academy of Sciences (Budapest). Glycogen was prepared from rabbit liver according to Mordoch et al. (1966). Other chemicals were purchased from Reanal (Budapest). Phosphorylase b was isolated from rabbit skeletal muscle (Fischer and Krebs, 1962) and was converted to phosphorylase a with rabbit muscle phosphorylase kinase and ATP or [y-32p]ATP (Krebs and Fischer, 1962). The specific radioactivity of 32p labeled phosphorylase a was 3 × 106Bq/mgprotein. Phosphorylated inhibitor-1 and nonphosphorylated inhibitor-2 were purified from rabbit skeletal muscle as described by Shenolikar and Ingebritsen (1984).
*To whom correspondence should be addressed. Abbreviations: Mops, 3-(N-morpholino)propanesulfonic acid; Tris, tris-(hydroxymethyl)-aminomethane;EDTA, ethylenediamine-tetraacetic acid. C.B.P. S7/4~-N
857
Animals and sample preparation Drosophila melanogaster (Canton-S strain) was reared on standard cornmeal medium at 25°C according to Ashburner and Thompson (1978). Eggs (~ 3 hr old) and first instar larvae (1 day old) were rinsed off the surface of the medium by a Drosophila ringer, containing 5 mM Mops, pH 6.8, 10 mM D-glucose, 128 mM NaC1 and 4.7 mM KC1, and were washed three times in the same solution. Third instar larvae (84 days old) and pupae
858
VIKTOR DOMBR~,DI et al. 100
80
60
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5
10
<
40 INHIBITOR-1
<
100
800
( U/ml )
100,
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°
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substrate and was carried out at 30°C. 20 #1 samples were withdrawn at appropriate time intervals (2-10 min), diluted 20-fold in a solution containing 40 mM ~-glycerophosphate, pH6.8, 2mM EDTA, 10mM 2-mercaptoethanol and 100 mM NaF, and the residual phosphorylase a activity was determined (Illingworth and Cori, 1953). The dephosphorylation reactions were linear up to 20% conversion of the substrate, accordingly, the homogenates were diluted with the homogenisation buffer so that no more than 20% of phosphorylase a was dephosphorylated within 10min. One unit of phosphorylase phosphatase liberates 1 nmol phosphate from the substrate within 1 min under these experimental conditions. The validity of phosphatase assay was proven by two facts: (i) 20 mM NaF, a well known inhibitor of phosphorylase phosphatase, was sufficient to block the interconversion of phosphorylase a (cf. Thoen et al., 1985). (ii) More than 95% of the radioactivity liberated from a2p labeled phosphorylase a was extracted as phosphomolybdate by organic solvent, indicating that inorganic phosphate and not phosphopeptide(s) was produced in the assay (Shenolikar and Ingebritsen, 1984). Inhibitors-I and -2 were assayed with the catalytic subunit of phosphatase-1 isolated from rabbit skeletal muscle as described by Shenolikar and Ingebritsen (1984). One unit of
2O
Vo ~ . ~
1
10 INHIBITOR-2
100
400
~= ~
~
0.6
(U/ml)
Fig. 1. Effect of rabbit muscle inhibitors-I and -2 on phosphorylase phosphatase activity of crude D. melanogaster imago extracts. The phosphatase activity of 10-fold diluted extracts was determined after preincubation in the presence or absence of the heat-stable inhibitor proteins. The activity without addition (0.4-0.6 U/ml) was taken as 100%. Each point represents the mean of three independent determinations; the standard deviation was less than + 6%.
04
0.2
0 20 :>
30
12
40
50
40
50
,~
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(1 day after pupation) were collected from the wall of the culture jar. Adult flies (2 weeks old) were harvested after quick anaesthesia with diethyletber. The brain of third instar larvae was dissected under a stereomicroscope. The neck of imagos was broken by mechanical agitation in liquid nitrogen and the heads were separated from other body parts by sequential seiving. All the above samples were stored in liquid nitrogen until homogenization. The samples were homogenized in 40 mM Tris buffer adjusted to pH 7.2 with 0.1 M HCI at room temperature and containing 2 mM EDTA, 10 mM 2-mercaptoethanol, 5 mM benzamidine, 1 mM o-phenantroline and 0.1 mM phenylmethylsulfonylfluoride by a motor-driven Teflon-glass Potter device with 25 strokes at 0°C. The homogenates were centrifuged at 10,000g (r~. 6cm) and 0°C for 10 min and supernatants were filtered through glass wool. Assays Protein phosphate activity was measured with rabbit skeletal muscle phosphorylase a that was shown to be a good substrate of insect phosphatases (Ashida and Wyatt, 1979; Hayakawa, 1985). The assay was based on the procedure of Brandt et al. (1975). The reaction mixture was composed of 100/~1 homogenate, 50 #1 40 mM Tris x HC1, pH 7.2, 2 mM EDTA, 10 mM 2-mercaptoethanol containing various additions and 100#1 25 U/ml rabbit muscle phosphorylase a and 12.5 mM caffeine dissolved in the same buffer. The reaction was started by the addition of the
1.0
03 o
0.8
a_
0.6
¢04 O
.=
20
f\
30
FRACTION
NUMBER
Fig. 2. Gel filtration of fruit-fly protein phosphatases. A 1.6 x 33 cm Sephacryl S-200 column was equilibrated and developed with 50 mM imidazole, pH 7.2, buffer containing 5 mM EDTA, 10 mM 2-mercaptoethanol, 0.1 M NaC1 and 10% glycerol at 6 _+2°C. The column was run at 10ml/hr flow rate and fractions of 1 ml were collected. 0.5 ml crude imago extract containing 8.2 mg protein (upper panel) or 0.5ml (NH4)2SO4, ethanol-treated extract containing 0.25 mg protein (lower panel) was applied onto the column and the phosphorylase phosphatase activity of the fractions was determined. Arrows indicate the elution position of marker proteins (molecular mass in kilodalton).
Protein phosphatases in an insect the inhibitors causes 50% inhibition of 20 mU phosphatase in the above assay system, The effect of inhibitors on Drosophila phosphatases was studied after 10min preincubation at 30°C with the diluted homogenates. Protein was measured by the dye-binding method of Bradford (1976). Bovine serum albumin obtained from Calbioehem (Los Angeles, CA) was used as standard and its concentration was checked spectrophotometrically (A~ = 6.5).
859
phosphatase (Ingebritsen et aL, 1980; Silberman et al., 1984) we assumed that the low molecular mass
phosphatase was composed of two different types of catalytic subunit and that the subunits could be separated by heparin-Sepharose chromatography (G-ergely et al., 1984). Indeed, two peaks of phosphatase activity were obtained after heparinSepharose chromatography (Fig. 3). The portion of phosphatase that did not bind to the affinity matrix was ..ot influenced by inhibitor-l, while the portion RESULTS bound to the column and eluted at 0.19 M NaCI was Inhibitor-sensitive and -insensitive protein phos- fully inactivated by the inhibitor protein. phatases The combination of (NH4)2SO4 and ethanol preInhibitors-1 and -2 isolated from rabbit skeletal cipitation with heparin-Sepharose chromatography muscle inactivated phosphorylase phosphatase in yielded a partially purified type 1 catalytic subunit crude extracts of adult Drosophila (Fig. 1). The preparation free of type 2 phosphatase. An 800--1000inhibition was concentration dependent; 800 U/ml fold purification was achieved by this simple proinhibitor- 1 or 400 U/ml inhibitor-2 caused practically cedure. total inactivation of the enzyme. The presence of inhibitor-insensitive phosphatase in the same extract was revealed by a so called Developmental study All of the above experiments were carried out with 'activating' treatment. Precipitation of the homogenate (6-8 mg/ml protein) by (NH4)2SO4 and ethanol samples obtained from the whole body of adult according to Brandt et al. (1975) enhanced phos- insects. The changes in phosphorylase phosphatase phorylase phosphatase activity to 127 _ 11% (N = 5) activity during the development of the flies was also of the original value. After activation only 60-70% of studied (Table 1). The specific activity of soluble the enzyme activity was inhibited by 1000U/ml phosphatase was the highest in early developmental inhibitor-l. The (NH4)2SO4 and ethanol treatment stages, i.e. in eggs and first instar larvae and was the converted larger molecular mass forms of phos- lowest in pupae. In all developmental stages the great majority of spontaneously active enzyme was sensiphatase into a form of Mr ~ 37,000 (Fig. 2.). tive to inhibitor-1. Two samples enriched in nervous Separation of two catalytic subunits tissue were also analysed. The larval brain and imago Based on analogies with the mammalian protein head contained higher specific activity of phosphatase
1-25 ~-
II
10
20 FRACTION
30
40
1
lI
°
50
NUMBER
Fig. 3. Affinity chromatography of D. melanogaster protein phosphatase catalytic subunits. A I x 29 cm heparin-Sepharose column was equilibrated with 50 mM imidazole, pH 7.2, buffer containing 5 mM EDTA and 10mM 2-mercaptoethanol at 6+2°C with 20ml/hr flow rate. 50ml of (NH4)2SO4, ethanol-treated fruit fly extract (0.35 mg/ml protein) dialysed against the column buffer was loaded on the column. The column was washed with the same buffer and eluted with a linear 0-0.5 M NaC1 gradient. The volume of fractions 1-18 was 5.1 ml and of fractions 19-52 was 1.6 ml. The fractions were assayed for phosphorylase phosphatase activity in the absence (O) and in the presence ( 0 ) of 1000U/ml inhibitor-l, and for protein ( x ). The figure is representative of five similar separations.
860
VIKTOR DOMBRId)I et al.
Table 1. Levels of protein phospohatases at various developmental stages of D. melanogaster Phosphorylase phosphatase activity Stage Total (U/mg protein) Type 1 (%) Egg 1.4 + 0.3 84 ± 8 First instar larva 1.4 ± 0.2 99 ± 4 Third instar larva 0.75 + 0.08 90 ± 5 Puparium 0.27 ± 0.05 83 ± 7 Imago 0.76 ± 0.17 96 ± 4 Brain of third 2.3 ± 0.7 60 ± 13 instar larva Head of imago 1.6 ± 0.5 70 ± 13 Means of 7-11 determinations + standard deviations are given. Whole bodies were used unless otherwise indicated. The specific activity of phosphorylase phosphatase was measured in crude extracts. The percentage of type 1 phosphatase corresponds to the activity inhibited by 800 U/ml heat stable inhibitor-1.
than the respective whole bodies and 30-40% of this activity was not affected by inhibitor-1 (Table 1). DISCUSSION
Rabbit muscle inhibitors-1 and -2 caused a dosedependent inactivation of Drosophila phosphorylase phosphatase. The inhibition proved to be selective. The catalytic subunit that was bound to heparinSepharose was totally inhibited while the unbound catalytic subunit was not influenced. It is known that heparin-Sepharose specifically adsorbs the catalytic subunit of type 1 phosphatase and does not interact with type 2 phosphatases (Gergely et al., 1984). The findings indicate that the classification of protein phosphatases originally designed for mammalian tissues (Ingebritsen and Cohen, 1983) can be extended to the realm of insects, and that the thermostable inhibitor proteins isolated from rabbit skeletal muscle can be widely used. Our results substantiate the classification of the two inhibitor-insensitive shrimp phosphatase as type 2 enzymes (Thoen et al., 1985) and will facilitate the detection of type 1 phosphatases in non-mammalian tissues. The fact that rabbit inhibitor proteins specifically interact with type 1 fruitfly phosphatase both in crude extracts and in a partially purified catalytic subunit preparation suggests that the regulation of type 1 phosphatase by the inhibitors must have been well conserved during evolution. Another similarity between the well-known mammalian and the here characterized insect phosphatases is the activation and dissociation of larger molecular mass enzyme forms into Mr ~ 37,000 catalytic subunits by (NH4)2SOq-ethanol treatment (Brandt et al., 1975). We found significant alterations in phosphorylase phosphatase activity during the development of Drosophila, however, in all stages type 1 phosphatase was predominant. Only in brain and head samples were considerable amounts of spontaneously active type 2 phosphatase present. These results are in agreement with the findings that many mammalian tissues contain type 2 phosphatase in a latent form (Di Salvo et al., 1984; Schlender and MeUgren, 1984; Erd6di et al., 1985) and that rabbit brain is a rich source of active type 2 p h o s p h o r y l a s e p h o s p h a t a s e (Ingebritsen et al., 1983). The c o o r d i n a t e d control o f type 1 p h o s p h a t a s e by inhibitor-1 is well d o c u m e n t e d (cf. Cohen, 1985). The p r e d o m i n a n c e of type 1 p h o s p h a t a s e in Droso-
phila makes it probable that the same mechanism can operate also in this insect. Acknowledgements--Thanks are due to Dr Ferenc Erd6di, Institute of Medical Chemistry, University School of Medicine, Debrecen, for the preparation of inhibitor-I and -2. The excellent technical assistance of Mrs. Margit Bir6 and Mrs. Agnes Csurg6 is acknowledged. This work was supported in part by the Hungarian Ministry of Health. Note added in proof. After submission of this manuscript we became aware of a work by S. Orgad, Y. Dudai and P. Cohen (Eur. J. Biochem., in press) in which these authors also detected both type 1 and type 2 protein phosphatases in Drosophila heads using heat-stable inhibitor-I and -2 from rabbit muscle. We thank Dr Philip Cohen for kindly sending us the manuscript before publication.
REFERENCES
Ashburner M. and Thompson Jr. J. N. (1978) The laboratory culture of Dorosphila. In The Genetics and Biology of Drosophila (Edited by Ashburner M. and Wright T. R. F.), Vol. 2, pp. 2-109. Academic Press, New York. Ashida M. and Wyatt O. R. (1979) Properties and activation of phosphorylase kinase from silkmoth fat body. Insect Biochem. 9, 403-409. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-253. Brandt H., Capulong Z. L. and Lee E. Y. C. (1975) Purification and properties of rabbit liver phosphorylase phosphatase. J. biol. Chem. 250, 8038-8044. Cohen P. (1985) The role of protein phosphorylation in the hormonal control of enzyme activity. Eur. J. Biochem. 151, 439-448. Di Salvo J., Gifford D. and Kokkinakis A. (1984) Modulation of aortic protein phosphatase activity by polylysine. Proc. Soc. exp. Biol. Med. 177, 24-32. Erdfdi F., Csortos Cs., Bot G. and Gergely P. (1985) Separation of rabbit liver latent and spontaneously active phosphorylase phosphatases by chromatography on heparin-Sepharose. Biochem, biophys. Res. Commun. 128, 705-712. Fischer E. H. and Krebs E. G. (1962) Muscle phosphorylase b. Meth. Enzymol. 5, 369-373. Gergely P., Erd6di F. and Bot G. (1984) Heparin inhibits the activity of protein phosphatase-1. FEBS Lett. 169, 45-48. Hayakawa Y. (1985) Activation mechanism of insect fat body phosphorylase by cold. Phosphorylase kinase, phosphatase and ATP level. Insect Biochem. 15, 123-128. lllingworth B. and Cori G. T. (1953) Crystalline muscle phosphorylase b. In Biochemical Preparations (Edited by Snell E. E.), Vol. 3, pp. 1-9. John Wiley, New York. Ingebritsen T. S. and Cohen P. (1983) Protein phosphatases: Properties and role in cellular regulation. Science 221, 331-338. Ingebritsen T. S., Foulkes J. G. and Cohen P. (1980) The broad specificity protein phosphatase from mammalian liver. Separation of the Mr ~ 35,000 catalytic subunit into two distinct enzymes. FEBS Lett. 119, 9-15. Ingebritsen T. S., Stewart A. A. and Cohen P. (1983) The protein phosphatases involved in cellular regulations6. Measurement of type-I and type-2 protein phosphatases in extracts of mammalian tissues; an assessment of their physiological roles. Eur. J. Biochem. 132, 297-307. Krebs E. G. and Fischer E. H. (1962) Phosphorylase kinase from rabbit skeletal muscle. Meth. Enzymol. 5, 373-376. Mordoch J., Krisman C. R. and Leloir U F. (1966) Further
Protein phosphatases in an insect studies on high molecular weight glycogen. Arch. Biochem. Biophys. 113, 265-272. Schlender K. K. and Mellgren R. L. (1984) Isolation of historic HI-stimulated phosphoprotein phosphatase from kidney and skeletal muscle. Proc. Soc. exp. Biol. Med. 177, 17-23. Shenolikar S. and Ingebritsen T. S. (1984) Protein (serine and threonine) phosphate phosphatases. Meth. Enzymol. 107, 102-129.
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Silberman S. R., Speth M., Nemani R., Ganapathi M. K., Dombrfidi V., Paris H. and Lee E. Y. C. (1984) Isolation and characterization of rabbit skeletal muscle protein phosphatases C-I and C-II. J. biol. Chem. 259, 2913-2922. Thoen C., Van Hove, Cohen P. and Slegers H. (1985) Identification of protein phosphatases dephosphorylating mRNP proteins from cryptobiotic gastruale of the brine shrimp A. salina. Biochem. biophys. Res. Commun. 131, 84-90.