Protein kinase C in Trypanosoma cruzi epimastigote forms: partial purification and characterization

Molecular and Biochemical Parasitology, 36 (1989) 11)1-108 Elsevier

101

MOI,BIO (11184

P r o t e i n k i n a s e C in Trypanosoma cruzi e p i m a s t i g o t e partial purification and c h a r a c t e r i z a t i o n

forms:

Maria L. G6mez t, Leonardo Erijman ~, Silvia Arauzo 2, H6ctor N. Torres ~ and Maria T. T611ez-lfi6n ~ Ilnstituto de Investigaciones en lngenieria Gen&ica ~, Biologia Molecular ~INGEBI), Facultad de ('ienctas Exacta~' v Naturales, Univer.~idad de Buenos Aires. Buenos Aires. Argentina. and : lnstituto de Inves'tigaciones Bioquimieas 'Fundacion ('ampomar'. Buenos Aires, Argentina

(Received 23 January 1999; accepted lfi March 198~,~)

A protein kinasc C activity from epimastigotc forms of Trypanosoma cruzi was characterized. Cvtosolic extracts were chromatographed on DEAE-ccllulose columns giving two peaks of kinasc activity which were clutcd at 0.1 and 0.15 M Na('l. The first activity peak requires Ca 2" and phosphatidylserine for activity. Further kmase purilication was performed by chromatography on phcnyl Scpharose colt, runs. In these columns the enzyme activity was adsorbed in the presence of Ca"" and cluted with a EGTAcontaining buffer. T. cruzi protein kinasc C activity prcfcrentiably phosphorylatcd histonc HI. It was stimulated by diacylglyccrol and phorbol myristatc acetate, and inhibited by polymyxin B and staurosporinc. After subcellular fractionation of epimastigote cells, the kinasc was found to be associated with microsomal and cvtosolic fractions. Key words: Trypanosoma cruzi: Protein kinase (7': Diacylglyccml: Phorbol ester; Staurosporinc

Introduction

Protein kinase C has a widespread distribution in higher eukaryotic organisms. It has been reported in several animal and plant tissues [1,2]. This enzyme activity is associated with receptor-mediated responses to some hormones, neurotransmitters and growth factors which require the mobilization of calcium ions for their actions. Participation of protein kinase C activity in the regulation of cell proliferation and differentiation is particularly important [3]. Correspondence address: Dr. M.T. T611ez-lfiOn, INGEBI, ()bligado 2490. 142g Buenos Aires, Argentina. .Vote: A preliminary report of this work has been been publishcd: Tellez-lfi6n, M.T.. Ulloa, R.M.. Gomcz. M.L.. Mcsri. E., and Torrcs. H.N (1988) Cyclic AMP-dependent and Ca:-phospholipid-activated protein kinasc activities in T. cruzi. XV Annual Meeting on Basic Research in Chagas' Disease, Caxarnbt], Brazil. Mcm. Inst. Oswaldo Cruz, Suppl. 83. l()g. Abbreviations:

PDS, phosphatidylscrinc, DG. diacylglycerol

Enzymatic activity requires Ca z- and phospholipids. Under physiological conditions, it is associated with membrane fractions, and can be activated by diacylglycerol (DG). This latter compound, and inositol-l,4,5-trisphosphate, are the products of phosphoinositide hydrolysis by phospholipase C. These compounds act as second messengers of the above mentioned receptor-mediated responses [4.5]. Trvpanosorna cruzi, the etiological agent of Chagas" disease, adapts to a variety of conditions imtx~sed by the insect vector and mammalian host environments during its life cycle. The adaptation involves important morphological, biochemical and proliferative changes, the control mechanisms of which are not well understood. The transition from the epimastigotc proliferative form to the trypomastigote, non-proliferative, infectious form has particular significance. This transition seems to be a consequence of the increase in cyclic AMP levels, another second messenger of rcccptor-mcdiated phenomena [6--8]. Work done in this laboratory indicates that cyclic AMP

1)166-6851,'89/$03.511 © 1989 Elsevier Science Publishers B.V. (Biomedical l)ivision)

1o2

specifically activates a protein kinase [9] and can be hydrolyzed by a phosphodiesterase activity under the control of the Ca2*-calmodulin complex [10]. These facts indicate that several control mechanisms involving second messenger functions are conserved during evolution from lower to higher eukaryotic organisms. In protozoa, the role of products of phosphoinositide breakdown by phospholipase C as second messengers, and enzyme effectors is completely unknown. However. preliminary work indicates the existence of a phospholipase C activity in 1". cruzi epimastigotes [11]. In addition, tumor promoters of the phorbol ester family, which are well known activators of mammalian protein kinase C activity, induce morphological changes in Trypanosomatidae cells [12,13]. As an extension of our work on the metabolism and role of second messengers in 7'. cruzi, this paper reports the presence, characteristics, partial purification and subcellular distribution of a protein kinase activity C in epimastigote forms of this parasite. Materials and Methods

Materials Polymyxin B, phenylmethanesulfonyl fluoride, leupeptin, trypsin inhibitor, benzamidine, bovine brain phosphatidylserine (PDS), DG (1,3-dioleilglycerol), phorbol myristate acetate (12-o-tetradecanoyl phorbol 13-acetate), E G T A , protamine, and histones HI (IIIS), VIS, VIIS, and VIIIS were from Sigma (St. Louis, MO, U.S.A.), Phenyl-Sepharose CL-4B was purchased from Pharmacia (Uppsala, Sweden), and DEAE-Cellulose (DE-52) and phosphocellulose paper (PS1) were from Whatman Chemical Separation (Clifton, N J, U.S.A.). Staurosporine was a gift of Ciba-Geigy (Basel, Switzerland), [32p]Pi and Omnifluor were from New England Nuclear (Boston, MA, U.S.A.), and [~-32p]ATP was prepared according to the method of Walseth and Johnson [14] modified by Gupa et al. [15]. Aprotinin (Trasylol) from Bayer (Leverkusen, F.R.G.) was a gift of Laboratorios Gador (Argentina). All other chemicals used were of reagent grade.

Cell cultures 7". cruzi epimastigote forms (Tulahuen 2 strain) were cultured for 7 days at 28°C, up to the late exponential phase, in monophasic axenic medium. All the components of this medium, including salt, hemin, peptone and tryptose and liver, brain, heart or ,,'east extracts were autoclaved before use for 15 min at 118°( [16]. TM

Enzyme purification ('rude extracts. (]'ells were collected by centrifugation at 1000 × g for 10 rain and washed three times with a 0.25 M sucrose solution in 5 mM KCI. Cells were homogenized in the same solution (10 ml g ~ of wet cells) with a Sorvall Ribi press operated at 34.5 MPA (5000 lb in 2) under an N2 atmosphere. Cell debris was discarded by centrifugation at 10(10 × g for 10 min. The supernatant, previously adjusted to 0.1 mM phenylmethanesulfonyl fluoride, 25 units ml ~ of Trasylol, (I.(ll~k leupeptin (w/v), 0.2 mg m1-1 trypsin inhibitor and 1 mM benzamidine was further centrifuged at 1(15000 × g for 6(I min. The supernatant fluid, referred to as 'soluble crude extract' and the sediment ('crude membrane fraction') were immediately processed to avoid proteolytic degradation. DEAE-celhdose chromatography of the 'soluble crude extract'. The 'crude extract" (300 ml; 1.1 mg protein ml i) was loaded on a DEAE-ccllulose column (13.8 x 2.7 cm) equilibrated with 20 mM Tris-HCl bufl'er, pH 7.4, containing 2 mM EDTA and 5 mM 2-mercaptoethanol (buffer A). The column was washed with 200 ml of buffer A and eluted with 30(I ml of a NaCI linear gradient (0 to 0.5 M) in the same buffer solution. Fractions of 5 ml were collected at a rate of 0.5 ml rain Those fractions exhibiting the highest protein kinase C activity which eluted at about 0.1 M NaCI, referred to as ' D E A E fraction', were pooled and subjected to chromatography on a phenyl-Sepharose column. PhenyI-Sepharose chromatography. Ca 2~-dependent adsorption of 1. cruzi protein kinase C activity to phenyl Sepharose CL-4B was performed according to Walsh et al. [17] with some modifications. The column (1.0 × 0.63 cm) was

IO3

equilibrated with buffer A containing 0.1 mM CaCI~. The ' D E A E fraction' (13 ml, (1.5 mg protein ml -~) was adjusted to 2 mM CaCI~ and 2 mM MgCI2, and loaded into the column. After washing with the equilibrium buffer until no absorbance at 280 nm was detected, and 30 ml of buffer A containing 0.5 M NaCI, the enzyme activity was eluted with buffer A containing 1 mM E G T A . Fractions eluting with this EGTA-containing buffer bearing protein kinase C activity were referred to as "phcnyI-Sepharose'. These fractions were used as enzyme source throughout this work.

DEAE-celhdose chromatography of the 'crude membrane fraction'. Pellets of the 'crude m e m brane fraction' were homogenized in buffcr A containing 1% Triton X-I(XI (v/v), 1 mM E G T A . 0.1 mM phenylmethanesulfonyl fluoride, 25 units ml-~ Trasylol, 0.01% Icupcptin (w/v), (1.2 mg ml -~ trypsin inhibitor and 1 mM bcnzamidine with a I)ouncc-type homogenizer and left to stand at 0°C for 60 min. The mixture was then centrifuged for 1 h at 105000 × g. The supernate (75 ml, 0.5 mg protein ml ~) was sublcctcd to chromatography on a DEAE-CclIulose column (7 × 2.7 cm) as described abovc. Wash and clution gradient volumes were both 200 ml. t'rotein kinase C activity assay Incubation mixtures contained 20 mM Tris-HCl buffer, ptl 7.4, 10 mM MgCI:, 5 mM NaF, 0.1 mg ml ' histone H1, and 50 txM [3,-32p]ATP (specific activity 500-1(X)0 cpm pmol -l) plus 1).5 mM E G T A or a mixture of 0.5 mM CaCI 2, 6(1 I-tg m l - 1 PDS and 3 ~g ml -~ D G (lipids were sonicated before adding to the mixtures). Total volume was 0.06 ml. Incubations were performed at 30°C for 15 min. Reactions were initiated by the addition of ['y-3-'P]ATP and stopped by spotting 40-1J,l aliquots of the reaction mixtures onto 2 x 2 cm square pieces of phosphocellulose paper, and immediately soaked in 75 mM H3PO 4, followed by three washes in the same solution [18]. The filters were dried and counted for radioactivity in an Omniftuor/toluene scintillation solution. Subcellular fractionation Cell pellets of T. cruzi epimastigotes (Tulahuen 2 or Tulahuen 0 strains) were resuspended

in 0.25 M sucrose solution containing 5 mM KCI (l(I ml g 2) of wet material) and lysed by three cycles of freezing and thawing. The homogenate was subjected to six successive centrifugations at 511(I x g for 15 min, 1(100 x g for 15 min, 500(I x g for 15 min, I I 000 x g for 3(1 min, 3(10(10 × g for 30 min, and 105000 x g for 60 min. Sediments from each ccntrifugation corresponded to "nuclear', 'flagellar', "mitochondrial', 'heavy lysosomal-glycosomar, 'light lysosomal" and 'microsomal'. All these fractions were resuspended in a (I.25 M sucrose solution containing 5 mM KC1 (l(I ml g ~ of wet material). These fractions and the last supernatant ('cytosolic fraction') were assayed for protein kinase C. Citrate synthase, hexokinase and malic enzyme activities were used as markers for the "mitochondrial', "glycosomal" and 'cytosolic" fractions respectively [19,20].

Analytical methods Protein was determined by the method of Lowry et al. [21]. Malic enzyme, citrate synthase, and hcxokinase were determined according to Cannata et al. [19,2(}]. Results

Protein kinase C purification from the "soluble crude extract'. Upon chromatography on D E A E cellulose of a T. cruzi "crude extract', two main protein kinase activity peaks (histone HI as substratc) were resolvcd (Fig. 1). These peaks eluted

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Fig. 1. Elution pattern of T. cruzi protein kinasc from a DEAE-cellulosc column. Protein kinasc activity was measured with histone tll in the presence of EGTA (:-;) or C a : ' , phosphatidylserine and diacylglycerol CO); ...... protein concentration: --, [NaCI]. Conditions as described in Materials and Methods.

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at NaCI concentrations of about O.l and 0.15 M NaCI. The first kinase activity peak was activated by Ca z" and phospholipids. This activity peak was designated protein kinase C. The second activity peak preferentially phosphorylated other histones and was not activated by Ca=- or phospholipids. The fractions corresponding to protein kinase (" activity were pooled and further purified by phcnyl Scpharose column chromatography (Fig. 2). Enzyme adsorption was performed in the presence of calcium and elution of enzyme activity was done with an E G T A - c o n t a i n i n g buffer.

Characterization of the protein kinase (" activio'. Enzyme activity was proportional to the incubation time up to ll) rain in the presence of Ca > and PDS. F.G'I'A addition completely abolished the activity (data not shown). As shown in Table 1, kinasc activity was dependent on the presence of Ca:" and PDS in the TABU-

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CaCI 2 [mMl Fig. 3. Protein k i n a s e C activity as a function o l ( ' a : " conOChre,lion. Protein k i n a s c C was a s s a y e d in the p r e s e n c e of E G T A (<:) or p h o s p h a t i d y l s c r i n c and diacylglycerol (o). C o n d i t i o n s as d e s c r i b e d in M a t e r i a l s and M e t h o d s .

incubation mixture. DG and phorbol myristate acetate, two well-known activators of mammalian protein kinases C, enhanced the enzyme activity determined in the presence of Ca 2+ and PDS. Under these conditions, kinase activity was roughly proportiomd to enzyme protein up to about 2 o-g protein per assay (data not shown). Fig. 3 shows the calcium dependence of T. cruzi protein kinase C activity. In the presence of PDS and DG the optimum total Ca z' concentration was 0.5 mM. and the K,, was about 0.15 raM. Requirements for PDS and D G are shown in Fig. 4. I n t h c presence of 3 ~ g m l ~ D G a n d 0 . 5 mM Ca z' half-maximal kinase activity was observed at 5 0 1 , g m l ] PDS(K,, = 3()lsgml ~,Fig. 4A); and in the presence of 60 ~g m l - t PDS and 0.5 mM Ca=' half-maximal stimulation was observed at about 3 gg ml -~ DG (K~ = 3.5 txg ml ~:

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Effect o l lipids, C a : " and E G ' I ' A

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Protein kinase activity ( p m o l m i n I (rag p r o t e i n ) t)

EGIA ( ' a e and diacylglyccrol P h o s p h a t i d y l s c r i n c and diacylglyccrol Ca-'" Ca-" and p h o s p h a t i d y l s e r i n c Ca r. . p h o s p h a t i d y l s c r i n c and p h o r b o l m y r i s t a t c a c e t a t e ( ' a " . p h o s p h a t i d y l s c r i n c and diacylglyccrol

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"Activities w e r e m e a s u r e d with O. 1 mg,'ml ~ histone t11. ( ' a :+. 0.5 raM: E G ' F A 0.5 raM; p h o s p h a t i d y l s e r i n e , 611 p.g'ml ~: diacvlglycerol 3 ~xg.ml ~ a n d p h o r b o l e s t e r , l(XI nM. O t h e r c o n d i t i o n s ,.,,ere as; d e t a i l e d u n d e r M a t e r i a l s and M e t h o d s .

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Fig. 4. Protein kinasc (" activity as a function of phosphatidvlserine (A) and diacylglycerol (B) concentrations. (A) mixtures contained ('a-' and diacylglyceml; (B) mixtures contamed Ca2" and phosphalidylserine. Conditions as described in Materials and Methods.

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Fig. 4B). Under the same conditions, phorbol myristate acetate could replace DG in the assay. Half maximal stimulation by the phorbol ester was usually observed at 25 nM (results not shown). Table II shows that histone H1 is the best phosphate acceptor for T. cruzi protein kinasc C activity. Phosphorylation rates with other protein substrates were below that observed in the absence of an exogenous phosphate acceptor. In the

Fig. 5. Effect of polymyxin B (o) and s t a u r o s p o r i n e (,~) on protein kinase (" activity. Enzyme activity was assayed in the presence of C a : " . phosphatidylserine and diacylglycerol. ('onditions ,ix described in Materials and Methods.

p r e s e n c e of Ca 2 ' , P D S and D G , the o p t i m a l histone H1 c o n c e n t r a t i o n was a b o u t 0.1 mg ml l H i g h e r c o n c e n t r a t i o n s lead to kinase inhibition. T h e K m for histone t l l was 2(1 ~g m l - I , with

T A B L E II Protein kinase (7 activity with different p h o s p h a t e acceptors Aceeptor

Concentration (mg"ml ~)

Protein kinase activity (pmoL'min ' (per mg protein) ~) with E G T A

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Activities ,*,ere m e a s u r e d in the presence of E G T A or a mixture of ('a z" , phosphatidylserine and diacylglycerol. O t h e r conditions were as detailed under Materials and Methods.

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Mic

Sol

Fig. ¢'~. DEAE-ccllulose column chromatography of a "crude membrane" fraction. Symbols were as in Fig. 1. Conditions as described in Materials and Methods.

either 60 or 120 p,g ml ~of PDS (data not shown). The effect of polymyxin B and staurosporine, two well-known inhibitors of brain protein kinase C activity, on the 7". cruzi enzyme was studied. As shown in Fig. 5. maximal effects of these two compounds were observed at 12 i.tM and 3 nM, respectively. Staurosporine clearly behaved as a partial inhibitor.

Purification and characteristics of the protein kitiase C activity associated to the 'crude membrane .fraction'. As shown below about 60-70% of the protein kinase C activity in epimastigote homogcnatcs from the Tulahuen 2 strain was found to bc associated with particulate fractions. This activity was solubilized bv extraction with a buffer containing 1% Triton X-100. After the extraction. the enzyme activity could bc purified by the same procedures uscd for the cnzymc present in the "soluble crude extract'. As shown in Fig. 6, the solubilized enzyme also cluted from a D E A E cellulose column at 0.1 M NaCI, and it was strongly activated by the Ca 2" P D S / D G mixture. In the presence of E G T A , enzyme activity was ncgligiblc. O t h e r properties were identical to those of the soluble form (results not shown). Subcelhdar distribution. Fig. 7 depicts the distribution of protein kinase C and m a r k e r enzymes in subccllular fractions obtained by differential ccntrifugation of h o m o g e n a t e s from two 7". cruzi strains: Tulahuen 2. the pathogenic strain used for kinase characterization, and the non-pathogenic Tulahuen 0.

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Fig. 7. Distribution of protein kinasc C activity and enzyme markers in different T. ( r u z i subccllular fractions. (A) Protcin kinase C activity in Tulahuen 2 (black bars) and Tulahucn l) (dashed bars). (B) Enzymc markers in Tulahuen 2 strain: ME. malic enzyme; CS. citrate synthasc; and IIK, hcxokinasc. N. 'nuclear': F, "flagcllar': Mit. 'mitochondrial': t11,, "heavy lysosomal-glycosomal'; LL. 'light lysosomal'; Mic, "microsomal': Sol, "cvtosolic' tractions.

In both strains, kinase activity was mainly located in 'microsomar and 'cytosolic' fractions. The distribution of marker enzymes in the different fractions was identical to that described by other authors [19,2(I]. Discussion

The results indicate the presence of protein kinasc C activity in T. cruzi epimastigotc cell extracts. Criteria to assert the authenticity of this enzyme activity are the following: (1) the enzyme requires Ca 2~ and phosphatidylscrinc for activity; (2) specificity for phosphate acceptors is characteristic of this type of phosphotransferase activ-

1117

ity; (3) the enzyme is activated by diacylglyccrol and phorbol myristate acetate, two well-known activators of mammalian protein kinase C [1,3,5]; and (4) it is inhibited by polymyxin B and staurosporine, which are inhibitors of animal protein kinasc C. Optimal concentrations for all these required compounds, activators and inhibitors arc almost identical to those reported for the mammalian protein kinasc C [22,23]. An interesting fact is that significant phosphorylation is catalyzed by T. cruzi protein kinase C preparations in the absence of an exogenous phosphatc acceptor (Table II). This phosphorylation may be due to the existence of an endogenous acccptor, which could be even the kinase itself. Autophosphorylation of animal protein kinasc C is a well-known phenomenon [24.25]. Another point that merits consideration is the subccllular distribution of this T. cruzi protein kinasc activity. As occurs in mammals, thc kinasc presents soluble and membrane-associated forms in cpimastigotc cells. Since the latter form is considcrcd to be the physiologically active one [1], it could be speculated that under the conditions by which epimastigote cells wcrc obtained for this work, at least 60% of the total protein kinase C is already activated. Moreover, the striking differences found in phosphorylation rates between the pathogenic Tulahucn 2 strain and the nonpathogenic Tulahucn 0 arc coincident with those observed in cyclic AMP-dependent protein kinase activity as previously described [9]. Additionally. some of the effects of phorbol esters reported in Trypanosomatidae, can be ex-

plained by the existence of this protein kinase C activity, which in animal cells is the receptor of such tumor promoter molecules [1,26], The occurrence of a protein kinasc C activity in T. cruzi cpimastigotes, and previous evidence on the control of T. cruzi cyclic nucleotide phosphodicsterase activity by homologous calmodulin [10] as well as the presencc of a cyclic AMP-dependent protein kinasc, suggest a similarity in the role and metabolism of second messengers between this parasite and mammalian cells. In addition, all this evidence argues in favor of the occurrence of specific receptor entities coupled to the enzyme sy,stems in the parasitc cell periphery responsible for the synthesis of the above-mentioned second messengers.

Acknowledgements We are indebted to Dr. Elsa L. Segura and Dr. AndrOs Ruiz from the lnstituto Nacional de Diagn6stico e Investigaci6n de la Enfermedad de Chagas, Buenos Aires for providing 7". cruzi Tulahuen 2 cultures, and to Dr. Juan J. Cazzulo, Instituto de Investigaciones Bioquimicas 'Fundaci6n Campomar" who provided T. cruzi Tulahuen 0 cultures. H.N.T. and M.T.T.-I. are Career Investigators of the Consejo National de lnvestigaciones Cientificas y T&nicas (Argentina). M.L.G. and L.E. are fellows of the Universidad de Buenos Aires. This work was supported in part by the World ltealth Organization Special Program for Research and Training in Tropical Diseases.

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60liveira. M.M., Antunes, A. and De Mello, F.G. (1984) Growth of Trypanosoma ~'ruzi epimastigotes controlled by shifts in cyclic AMP mediated by adrenergic ligands. Mol. Biochcm. Parasitol. 11,283-292. 7 RangeI-Aldao, R., AIIcnde, O., Triana, F., Piras, R., Hcnriquez, D. and Piras, M. (1987) Possible roles of cAMP in the differentiation of Trvpanosoma cruzi. Mol. Biochcm. Parasitol. 22, 39-43. 8 Gonzalcs-Perdomo, M., Romcro, P. and Goldenbcrg, S. (1988) Cyclic AMP and adenylate cydase activators slimulatc Trypanosoma cruzi differentiation. Exp. Parasitol. 66, 205-212. 9 Ulloa, R.M., Mesri, E., Esteva, M.. Torres, H.N. and l'ellez-lfi6n, M.T. (1988) Cyclic AMP-dependent protein

l()~

1()

II

12

13

14

15

16

17

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