The cAMP-binding proteins of Leishmania are not the regulatory subunits of cAMP-dependent protein kinase

Comparitive Biochemistry and Physiology Part B 130 Ž2001. 217᎐226

The cAMP-binding proteins of Leishmania are not the regulatory subunits of cAMP-dependent protein kinase Chandana Banerjee 1, Dwijen Sarkar U Leishmania Di¨ ision and Department of Cell Biology, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta 700 032, India Received 15 March 2001; received in revised form 4 June 2001; accepted 11 June 2001

Abstract The most commonly used method to determine the cAMP binding activity in cytosolic extracts of promastigotes of Leishmania spp. underestimated by approximately 11.5-fold the total amount of w 3 HxcAMP bound, when compared with results obtained by the modified Millipore filter technique. Three cAMP-binding proteins ŽBPI, BPII and BPIII. were partially purified and characterized. The native molecular masses of BPI, BPII and BPIII were estimated to be 105, 155 and 145 kDa, respectively. The binding of w 3 HxcAMP to these proteins was affected to different extents by several cAMP analogues. Antibodies directed against the types I and II regulatory subunits of PKA did not cross-react with the leishmanial extract. Photoaffinity labeling of the cytosolic extracts with 8-N3-w 32 PxcAMP specifically labeled a band of Mr 116 000 and a band of Mr 80 000 partially saturable by cAMP. From these results, it is concluded that the leishmanial cAMP-binding proteins appear to belong to a different class distinct from the regulatory subunits of cAMP-dependent protein kinases. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Cyclic AMP-binding protein; Promastigote; Leishmania; Visceral leishmaniasis; Kala-azar; Cyclic AMP; Azido cyclic AMP; Cyclic AMP-dependent protein kinase; Regulatory subunit of cyclic AMP-dependent protein kinase

1. Introduction Kinetoplastid protozoa of the genus Leishmania are of global importance as parasites, causing a spectrum of human diseases, the leishmaniasis. In India, the disease caused by Leishmania dono¨ ani is known as visceral leishmaniasis Žor kalaU

Corresponding author. Tel.: q91-33-413-6793r3491 ext. 135; fax: q91-33-473-5197. E-mail address: [email protected] ŽD. Sarkar.. 1 Present address: Department of Physiology, Vidyasagar College, Calcutta, India.

azar.. Leishmania undergoes a digenic life cycle. The flagellated promastigotervector form presumably senses a host biochemical signal after internalization into macrophages and transforms rapidly to an aflagellated amastigote form that escapes the host defense mechanism and initiates pathological conditions. Eukaryotic cells are highly regulated entities, most regulation being mediated directly or indirectly by covalent changes in proteins. The most common covalent means of regulating protein activity is by the phosphorylationrdephosphorylation mechanism mediated by protein kinasesrprotein phosphatases. The subse-

1532-0456r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 1 . 0 0 2 3 2 - 0

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quent covalent modification of target proteins and the associated changes in their function account for the physiological responses. Leishmania, a member of the Trypanosomatid family, possess components of the Ca2q ŽSarkar and Bhaduri, 1995; Banerjee et al., 1999. and cAMP signaling pathway, e.g. adenylate cyclase ŽSanchez et al., 1995., cAMP phosphodiesterase ŽRascon et al., 2000.. Although several independent kinases are reported ŽDas et al., 1986; Banerjee and Sarkar, 1990, 1992., it has been impossible to-date to demonstrate any messenger-dependent kinase in Leishmania spp. On the contrary, several kinases, including protein kinase C ŽKeith et al., 1990., Ca2qrCaM dependent kinase ŽOgueta et al., 1996. and cAMP-dependent protein kinase ŽPKA. ŽOchatt et al., 1993., have been isolated from a highly related species, T. cruzi. PKA is the most conserved and studied messenger-dependent kinase in the higher eukaryotic system due to its known important regulatory role in different cellular processes ŽTaylor et al., 1992.. We have been able to purify a protein kinase similar to the catalytic subunit of PKA ŽBanerjee and Sarkar, 1992.. Recently, a specific gene, clpk2, existing as a single copy in Leishmania dono¨ ani and showing homology to other eukaryotic PKA, has been demonstrated ŽSimon-Tov et al., 1996.. PKA expression has been found to be: restricted to promastigote stage; temperature sensitive; and post-transcriptionally regulated. PKA usually exists as an inactive holoenzyme consisting of two regulatory dimer ŽR 2 . and two catalytic subunits ŽC.. Upon binding of cAMP to R 2 dimer, the active C subunit is released. The free C subunit phosphorylates a number of cytosolic and nuclear proteins, leading to various physiological changes. In many eukaryotic cells, differentiation and development are not always associated with coordinate expression of the C and R subunits ŽSpaulding, 1993.. Elevated cAMP may often cause elevated levels of the C subunit with basal levels of the R subunit. As PKA has never been demonstrated without the presence of its corresponding regulatory subunit, we attempted in this study to use different biochemical approaches to study the several cAMP-binding proteins and look for the possible presence of the leishmanial PKA regulatory subunit.

2. Materials and methods w ␥- 32 Px-ATP Ž3000 Cirmmol. and w 125 Ix-NaI Ž90 mCirml. were purchased from Bhaba Atomic Research Center, India. w 3 H xcAMP Ž41.7 Cirmmol. was obtained from Amersham International Ltd, UK. 8-N3-w 32 PxcAMP Ž61.2 Cirmmol. was from ICN Chemicals, USA, 0.45-␮m nitrocellulose filter papers ŽHAWP. were from Millipore, USA. DEAE-cellulose and glass-fiber filter paper ŽGFrC. was obtained from Whatman. Molecular weight markers were from Boehringer Mannheim, Germany. Freund’s complete and incomplete adjuvants were from Difco Laboratories, USA. Sephacryl S-200 was obtained from Pharmacia, Sweden. All other chemicals were analytical grade and were purchased from E. Merck, Germany. 2.1. Source and growth of Leishmania promastigotes P ro m a stig o te s o f L eish m a n ia sp p . ŽMHOMrINr78rUR6., an Indian strain, highly subpassaged in modified Ray’s blood agar slants ŽRay, 1932., were grown at 24᎐26⬚C for 72 h Žlate exponential phase.. The cells were then harvested, washed three times in isotonic, cold phosphate-buffered saline, pH 7.4 containing 0.5 mM PMSF, 1 mM iodoacetic acid and 5 mM benzamidine᎐HCl and kept at y20⬚C until use. 2.2. Cyclic [ 3 H]AMP binding assay Cyclic w 3 HxAMP binding assays were performed either by the method of Gilman Ž1970. ŽMethod 1 in Table 1. or of Doskeland and Ueland Ž1977. ŽMethod 2 in Table 1.. The binding of w 3 HxcAMP was assayed at 4⬚C for 1 h with 1 ␮M w 3 HxcAMP Ž41.7 Cirmmol., in 100 ␮l of 50 mM potassium phosphate buffer, pH 7.1, 5 mM EDTA and 1 mgrml of bovine serum albumin and the appropriate amounts of proteins. After incubation, the reaction was terminated either by: addition of 1 ml of ice-cold 50 mM potassium phosphate buffer, pH 7.1, and filtration on Millipore filters with three 5-ml washings of the above buffer ŽMethod 1 in Table 1.; or precipitation of the w 3 HxcAMP᎐receptor complex with 1 ml of ice-cold 80% saturated ammonium sulfate followed by filtration on Millipore filters with two

C. Banerjee, D. Sarkar r Comparati¨ e Biochemistry and Physiology Part C 130 (2001) 217᎐226 Table 1 Specific w 3 HxcAMP binding activity of cytosolic preparation from Leishmania and 40 000 = g supernatant from bovine heart muscle Assay

L. dono¨ ani Bovine heart muscle

w3 HxcAMP binding Žpmolrmg protein. Method 1

Method 2

1.60 6.20

18.75 13.60

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mogenate was centrifuged at 100 000 = g for 90 min and the supernatant used for further purification.

Ratio 2r1

11.50 2.19

Samples of 100 000 = g cytosolic extract of Leishmania Ž50 ␮g. and 40 000 = g supernatant fraction of bovine heart muscle Ž30 ␮g. were assayed for w 3 HxcAMP binding by the methods of Gilman Ž1970. ŽMethod 1. and Doskeland and Ueland Ž1977. ŽMethod 2.. The ratio 2r1 represents the value given by dividing the results obtained by Method 2 over Method 1. The data are representative of three experiments.

3-ml washings of 60% saturated ammonium sulfate ŽMethod 2 in Table 1.. In both cases, the radioactive wet filters were immersed in 1 ml of 1% SDS and counted by liquid scintillation in 7 ml of a scintillation mixture containing 0.5% Žwrv. omnifluor and 33% Žvrv. Triton X-100 in toluene. Non-specific binding of w 3 HxcAMP was subtracted in each case by incubating a tube with 100 ␮M unlabelled cAMP.

2.4.2. DEAE-cellulose column The pH and conductivity of the 100 000 = g supernatant was adjusted to the starting buffer and applied onto a DEAE-cellulose column Ž3 = 15 cm. equilibrated in Buffer 1. The column was eluted with a 500-ml linear NaCl gradient from 0 to 500 mM in Buffer 1. Each fraction from the DEAE-cellulose column ŽFig. 1. was measured for w 3 HxcAMP binding, as well as phosphotransferase activity in the presence and absence of cAMP, using the well-known cAMP-dependent protein kinase substrate, protamine. Protein kinase activity eluted between 150 and 210 mM NaCl wThis enzyme has already been described ŽBanerjee and Sarkar, 1990..x Three peaks of cAMP-binding proteins were also obtained. Fractions 10᎐13, 16᎐18 and 24᎐30 were concentrated separately with an Amicon nitrogen pressure cell using a PM-30 membrane. They are designated as BPI, BPII and BPIII, in order of their elution from the DEAE-cellulose column.

2.3. Protein kinase assay Protein kinase activity was assayed as described earlier, using either protamine as the phosphate acceptor ŽBanerjee and Sarkar, 1990. or kemptide as substrate ŽBanerjee and Sarkar, 1992.. 2.4. Partial purification of the cAMP-binding proteins Unless otherwise stated all the operations were carried out at 2᎐4⬚C. 2.4.1. Preparation of cell extract Frozen packed cells Žapprox. 10 ml. were suspended in 50 ml of 10 mM Tris᎐HCl buffer, pH 7.2, containing 1 mM EGTA, 1 mM iodoacetic acid, 5 mM benzamidine HCl, 0.5 mM PMSF, 0.02% sodium azide and 10% Žvrv. glycerol ŽBuffer 1.. The cells were disrupted by three cycles of freezerthawing and the resulting suspension was homogenized and sonicated four times for 20 s on a Labsonic 2000 sonifier, with a 1-min interval between each sonication. The ho-

Fig. 1. DEAE-cellulose chromatography of Leishmania protein kinase and cAMP-binding proteins. A 100 000 = g supernatant from Leishmania extract was adsorbed onto a DEAEcellulose column and eluted with 500 ml of a linear Ž0᎐0.5 M. NaCl gradient in Buffer 1, pH 7.2. Fractions of 10 ml were collected. Aliquots of 25 ␮l from each fraction were assayed for protein kinase activity in the presence Ž--^--. or absence Ž--'--. of 10 ␮M cAMP. An additional aliquot Ž250 ␮l. was assayed for w 3 HxcAMP binding activity Ž--䢇--.. NaCl concentration is denoted by --`--.

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2.4.3. Sephacryl S-200 gel filtration Each of the separately concentrated fractions was applied individually to a Ž1.5= 10.4 cm. Sephacryl S-200 column pre-equilibrated with 50 mM Tris᎐HCl buffer, pH 7.2, containing 150 mM NaCl. The samples were eluted with the same buffer. The tubes containing the major cAMPbinding activity were pooled and concentrated separately with an Amicon PM-30 membrane and used as the source of partially purified cAMPbinding proteins in relevant experiments. 2.5. Preparation of PKI, PKII, RI and RII subunits of cAMP-dependent protein kinase Fresh bovine skeletal and heart muscle was obtained from the local slaughterhouse Ž800 g.. Highly purified RI subunits were prepared from bovine skeletal muscle and RII subunits were prepared from bovine heart muscle by a combination of the methods of Erlichman et al. Ž1980. and of Weber and Hilz Ž1978.. The tissues were homogenized in three volumes of 10 mM potassium phosphate buffer, pH 6.8, 20 mM benzamidine HCl, 5 mM ␤-mercaptoethanol and 4 mM EDTA ŽBuffer A. at 4⬚C. The cytosol was isolated by centrifugation at 40 000 = g for 15 min. The pH was adjusted to 6.8 and the conductivity of the buffer was adjusted to match the homogenization medium. The cytosol was applied to a column of DEAE-cellulose equilibrated with buffer A. The column was washed with two column volumes of Buffer A and then developed with a linear gradient of NaCl Ž0᎐0.3 M. in the same buffer. Fractions eluting between 0.03 and 0.08 M NaCl ŽPKI. and 0.14 and 0.2 M NaCl ŽPKII. were pooled separately, concentrated by precipitation with 70% ŽNH 4 . 2 SO4 , dialyzed and applied to a 8-Ž6amino hexyl.amino᎐cAMP Sepharose 4B column. RI and RII subunits were eluted from the affinity column either by Buffer A containing 10 mM cAMP or 6 M urea. The eluted RI and RII were rapidly passed through Sephadex G 25 and dialyzed extensively against Buffer A containing 50% glycerol to remove free cAMP or urea and for concentrating the preparations. The urea-eluted RI and RII were used for the relevant experiments. The subunit preparations were homogeneous, as judged by electrophoresis on SDS polyacrylamide gels.

2.6. Preparation of antisera against bo¨ ine muscle RI and bo¨ ine heart RII subunits of cAMPdependent protein kinase Antisera to highly purified muscle RI and heart RII were prepared in New Zealand white rabbits. Antisera against RI and RII were raised in separate rabbits. A sample of 1 mg of the R subunits Ž0.5᎐0.6 ml in Buffer A. was emulsified in an equal volume of Freund’s complete adjuvant. Half of the emulsified sample was injected intradermally at multiple sites on the back of the rabbits. The rest of the sample was injected subcutaneously. The rabbits were given three booster injections subcutaneously in incomplete adjuvant at 10-day intervals. Approximately 7᎐10 days after the final injection, anti RI and anti RII titers reached maximum levels, which was maintained for the next 3᎐4 weeks. 2.7. Western blot analysis Samples of 150 ␮g of 100 000 = g supernatant from Leishmania and 80 ␮g each of PKI and PKII were subjected to electrophoresis in 0.1% SDS, 10% polyacrylamide gels until the dye front reached the bottom of the gel. The resolved polypeptides were transferred electrophoretically to nitrocellulose paper Ž0.45 ␮m, Schleicher and Schuell. by the Western blotting procedure described by Burnett Ž1981.. The details have been described elsewhere ŽSanyal et al., 1994.. 2.8. Radioiodination of Protein A w 125 Ix-labeled protein A Ž3 = 10 8 cpmr␮g. was prepared by the procedure of Schubart and Fields Ž1984.. 2.9. Photoaffinity labeling Covalent incorporation of 8-N3w 32 PxcAMP into the regulatory subunit of PKII was carried out as described before ŽRubin et al., 1979.. Indicated amounts of 100 000 = g supernatant were incubated in duplicate for 45 min at 4⬚C in Limbro plastic microwells in the presence of 50 mM potassium phosphate buffer, pH 7.1, and 1 ␮M 8-N3-w 32 PxcAMP Ž61.2 Cirmmol.. After incubation, the wells were irradiated for 10 min with a Mineralight ultraviolet lamp at 254 nm and a

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distance of 7 cm Žintensity 1.37 mWrcm2 .. After irradiation, one set of samples was subjected to SDS polyacrylamide electrophoresis in 1.2-mmthick slab gels Ž10% polyacrylamide. at a constant current of 25 mA ŽLaemmli, 1970.. After electrophoresis, the gels were stained for protein with coomassie brilliant blue R-250 ŽRubin et al., 1979., dried and autoradiographed at y70⬚C with Kodak XAR-5 film in the presence of Quanta III intensifying screen. A 1-ml aliquot of 80% saturated ammonium sulfate was added to the remaining set of tubes and subsequently filtered through Millipore filters, as described for w 3 HxcAMP binding, except that an additional wash with 5% TCA was carried out to eliminate nonspecific binding. Non-specific binding of 8-N3w 32 PxcAMP was subtracted by adding 100 ␮M cAMP to the incubation mixture prior to ultraviolet irradiation. 2.10. Sodium dodecyl sulfate polyacrylamide gel electrophoresis Electrophoresis in 10% polyacrylamide slab gels containing 0.1% sodium dodecyl sulfate was carried out by the method of Laemmli Ž1970.. Gels were fixed, stained and destained according to Fairbanks et al . Ž1971.. For autoradiography, dried gels were exposed to Kodak XAR-5 film at y70⬚C in the presence of Quanta III intensifying screen. Films were developed for 5 min and fixed for 8 min, as suggested by the manufacturer.

3. Results and discussion Leishmanial cytosolic extracts were assayed for the presence of cAMP-binding activity by two well-established methods. It can be seen from Table 1 that although leishmanial cytosol contained cAMP-binding activity, it was underestimated by 11.5-fold when measured by the method of Gilman Ž1970. ŽMethod 1. as compared with the modified version described by Doskeland and Ueland Ž1977. ŽMethod 2.. However, only a twofold difference in activity was observed with the 40 000 = g supernatant fraction of bovine heart muscle. This difference observed for the total w 3 HxcAMP binding activity of bovine cardiac muscle could be explained by a partial loss on the Millipore filters of the cAMP bound to its

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cAMP-binding proteins. Such losses did not occur when the cAMP-binding protein᎐cAMP complex was precipitated with ammonium sulfate prior to filtration on Millipore membrane, as performed by Doskeland and Ueland Ž1977.. In the case of Leishmania, most of the cAMP bound to its cAMP-binding proteins may be lost in the unmodified Millipore filter assay. Alternatively, the Millipore filters may not retain some of these cAMP-binding proteins. Hence, it seems that the cAMP-binding property of the leishmanial extract was not similar to that of the mammalian counterpart. Due to the high cAMP-binding activity obtained by Method 2, all subsequent cAMP binding assays with the leishmanial extract were carried out by this method. As the leishmanial cytosolic extract possesses reasonable cAMP-binding activity, attempts were made to partially purify and characterize the cAMP-binding protein. As mentioned in the Section 2, the 100 000 = g supernatant of the leishmanial extract was applied onto a DEAE-cellulose column and the bound proteins were eluted with buffer containing a linear gradient of 0᎐500 mM NaCl ŽFig. 1.. The fractions were measured for w 3 HxcAMP binding, as well as phosphotransferase activity in the presence and absence of cAMP, using the well-known cAMP-dependent protein kinase substrate, protamine. One peak corresponding to cAMP-independent protein kinase activity was present ŽFig. 1., eluting at approximately 170᎐180 mM NaCl. This protein kinase phosphorylates exogenous substrate in the following order: protamine ) kemptide) mixed histones ) casein ) phosvitin ŽBanerjee and Sarkar, 1990.. Three peaks corresponding to cAMP-binding proteins were also obtained. These cAMP-binding proteins were not associated with any phosphotransferase activity in the presence or absence of cAMP ŽFig. 1.. They were designated as BPI, BPII and BPIII in the order of their elution from DEAE-cellulose column. These three fractions of cAMP-binding proteins were further purified separately through a Sephacryl S-200 column. Approximately 420 ␮g of BPI, 630 ␮g of BPII and 1.95 mg of BPIII were obtained, having a total cAMP-binding activity of 6.3, 100, and 92 pmol, respectively, from 10 ml of packed cells. The native molecular masses of the three binding proteins were estimated to be 105, 155 and 145 kDa, respectively, according to their elution from the calibrated Sephacryl S-200 column

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Fig. 2. Native molecular weight determination of the cAMP binding proteins from a calibrated Sephacryl S-200 column. Marker proteins used are indicated in the figure. The equilibrium distribution coefficients Ž K av . were plotted against log molecular weight Ž Mr .. The Mr of BPI, BPII and BPIII were estimated to be 105 000, 155 000 and 145 000, respectively.

ŽFig. 2.. Further purification of the cAMP-binding proteins by conventional affinity chromatography on cAMP agarose was not successful. This led us to further characterize the binding proteins in a partially purified form. Mixing the partially purified cAMP-binding proteins and the purified 34kDa leishmanial protein kinase, which resembles the C subunit ŽBanerjee and Sarkar, 1992., did not have any effect on the latter enzyme Ždata not shown., confirming that the partially purified

cAMP-binding proteins were not the regulatory subunit of cAMP-dependent protein kinase. The specificity of the cAMP-binding proteins from Leishmania is shown in Table 2. It can be observed that a 50-fold excess of non-radioactive cAMP could inhibit 89% of the binding of w 3 HxcAMP to BPIII, whereas it could inhibit the binding to BPI and BPII by only 63 and 78%, respectively. cGMP could inhibit only 42, 49 and 27% of w 3 HxcAMP binding to BPI, BPII and BPIII, respectively. There is a striking difference between BPIII and the other two binding proteins. BPIII was found to be much more specific for cAMP binding, as the adenine derivatives could not appreciably displace the bound w 3 HxcAMP. This difference in the binding of adenine derivatives to BPI and BPII compared to that reported for the regulatory subunits of cAMP-dependent protein kinase, suggests that these two binding proteins from Leishmania are not identical to the regulatory subunits. The C8 substituted analogues of cAMP, such as 8-bromo-cAMP, have been shown to be highly selective for one of the two cAMP-binding sites Žsite 1. which are present in equal amounts in both type I and type II regulatory subunits of cAMP-dependent protein kinases ŽRannels and Corbin, 1983.. Thus, the effect of 8-Bromo-cAMP on purified type II regulatory subunit from bovine heart and on the three cAMP binding proteins from Leishmania was studied. The w 3 HxcAMP-binding experiments shown in Fig. 3 revealed a very different behavior of the cAMP-binding proteins from Leishmania with respect to the regulatory subunit of cAMP-dependent protein kinase. Al-

Table 2 Specificity of w 3 HxcAMP binding by leishmanial cAMP-binding proteins Analogue

None cAMP cGMP AMP ADP ATP Adenosine

BPI

BPII

BPIII

pmol bound

Inhibition Ž%.

pmol bound

Inhibition Ž%.

pmol bound

Inhibition Ž%.

2.6 0.97 0.5 0.4 0.4 0.6 0.24

᎐ 63 42 85 85 72 91

1.7 0.49 0.87 0.41 0.24 0.58 0.44

᎐ 78 49 76 86 66 74

2.2 0.24 0.67 1.9 1.9 1.54 2.2

᎐ 89 70 14 14 30 0

To measure the specificity of cAMP binding, Amicon concentrated BPI, BPII and BPIII from DEAE-cellulose column as described in Section 2 were employed. Each assay contained 1 ␮M w 3 HxcAMP, and where indicated, 50 ␮M of the analogue. The results are representative of three experiments carried out in duplicate.

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Fig. 3. Binding competition of cAMP and 8-bromo-cAMP with w 3 HxcAMP for the regulatory subunit of cAMP-dependent protein kinaseŽRII. and the cAMP-binding proteins BPI, BPII and BPIII from Leishmania. Pure RII Ž60 ng. or partially purified ŽSephacryl S-200 elutes. cAMP-binding proteins of Leishmania Ž60 ␮g. were subjected to w 3 HxcAMP binding as described in Section 2 in the presence of the indicated concentrations of cAMP ŽPanel a. and 8-bromo-cAMP ŽPanel b.. Ž----䢇----., Ž-`-., Ž-I-. and Ž-^-. represent RII, BPI, BPII and BPIII, respectively, in Panel a. Ž----`----., Ž-䢇-., Ž-B-. and Ž-'-. represent RII, BPI, BPII and BPIII, respectively, in Panel b.

though reasonable specificity for cAMP was displayed by all the proteins, 8-bromo-cAMP, which was the most effective analogue of cAMP for the regulatory subunit, had practically no effect in inhibiting the binding of w 3 HxcAMP to the cAMP-binding proteins from Leishmania. An equal concentration of this analogue inhibited more than 90% of the binding of w 3 HxcAMP to RII. From these results, it appears that the leishmanial cAMP-binding proteins are not similar to the regulatory subunit of cAMP-dependent protein kinase. Several trypanosomatid proteins cross-react with antibodies directed against higher eukaryotic counterparts. Polyclonal antibodies directed against type I and type II regulatory subunits and the catalytic subunit from bovine heart crossreacted with regulatory and catalytic subunits of T. cruzi ŽOchatt et al., 1993.. Fig. 4 shows the Western blot analysis of the 100 000 = g supernatant fraction of Leishmania and partially purified type I and type II regulatory subunits of cAMP-dependent protein kinase using polyclonal antibodies directed against type I and type II regulatory subunits. Anti Žtype I regulatory subunit. immunoglobulin and anti Žtype II regulatory

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subunit . immunoglobulin recognized type I and type II regulatory subunit from PKI and PKII Žlanes 4 and 5., but failed to cross-react against the 100 000 = g supernatant fraction from Leishmania Žlanes 1 and 2.. The experiment was repeated with the cAMP-binding fractions from the Sephacryl column, but no cross-reactivity was detected Ždata not shown.. This clearly indicates that unlike T. cruzi, the cAMP-binding proteins of Leishmania are not the regulatory subunit of cAMP-dependent protein kinase. We then used the photoaffinity analogue of cAMP, 8-N3w 32 PxcAMP ŽHaley, 1977; Walter et al., 1977. to identify the cAMP-binding proteins of Leishmania at the molecular level. This experiment allowed us to compare such proteins with that of bovine heart muscle. When the 100 000 = g supernatant fraction from Leishmania was exposed to covalent incorporation of 8-N3-w 32 PxcAMP at increasing concentrations up to 1.5 ␮M of the photoaffinity analogue, only one protein band of Mr 116 000 was specifically labeled and saturated at a concentration of 1 ␮M Ždata not shown.. The leishmanial cAMP-binding proteins were observed to have much higher K d values for 8-N3-

Fig. 4. Western blot analysis of 100 000 = g supernatant of Leishmania and bovine PKI and PKII. Samples of 150 ␮g of 100 000 = g supernatant of Leishmania Žlanes 1᎐3. and 80 ␮g of bovine PKI and PKII Žlanes 4᎐5. were resolved by electrophoresis on 10% Žwrv. polyacrylamide gels, transferred to nitrocellulose and probed with the indicated antibodies. Lane 1 was treated with preimmune serum. Lanes 2 and 4 were treated with anti-RI antiserum. Lanes 3 and 5 were treated with anti-RII antiserum. Molecular weight standards were as indicated.

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w 32 PxcAMP compared to that obtained using w 3 HxcAMP in the modified Millipore filtration method. The high K d values for 8-N3-w 32 PxcAMP might reflect a much lower affinity of the leishmanial cAMP-binding proteins for the cAMP analogue with respect to other corresponding proteins of many species studied ŽWalter et al., 1977; Juliani and Klein, 1981; Sy and Roselle, 1981; Uno and Ishikawa, 1981; Rangel-Aldao et al., 1983.. Fig. 5 shows an autoradiogram, which confirms the different nature of the cAMP-binding proteins of Leishmania with respect to bovine cardiac muscle when such proteins were labeled with 8-N3-w 32 PxcAMP. In this case, to obtain the maximum labeling we employed a 1-␮M concentration of the photoaffinity probe, which we have found to give maximum labeling into the Mr-116 000 protein band. As we can see, bovine cardiac muscle PKII ŽFig. 5, lanes 1and 2. showed two bands specifically labeled. The Mr 55 000 corresponds to the dephosphorylated form of the regulatory subunit of type II cAMP-dependent protein kinase and the Mr 40 000 may be the proteolyzed product of the Mr 55 000. It is interesting to note that although we have found three distinct cAMPbinding proteins from the 100 000 = g supernatant fraction from Leishmania through a DEAE-cellulose column, photoaffinity labeling of this supernatant with 8-N3-w 32 PxcAMP showed only one band of Mr 116 000 specifically labeled ŽFig. 5, lanes 3 and 4.. Another band of Mr 80 000 was found to incorporate a considerable amount of the photoaffinity analogue, only 70% of which could be swamped out with excess cAMP. The massive non-specific incorporation of the photoaffinity analogue of cAMP observed in the present study is in agreement with the earlier observations of Rangel-Aldao et al. Ž1983. and Juliani and Klein Ž1981. in their studies with T. cruzi and D. discoidium, respectively. Apparently, such effects in photoaffinity labeling occur when the concentration of 8-N3-w 32 PxcAMP is used higher than 1 ␮M in order to label a specific protein, which is either present in low concentration or has a very low affinity for the photoaffinity probe, as in the case of cAMP-binding protein from Leishmania. Most of the cAMP-binding proteins present in Leishmania has been found to be cytosolic, although we have found a certain amount of cAMP-binding activity in the membrane fraction

Fig. 5. Photoaffinity incorporation of 8-N3 -w 32 PxcAMP into 100 000 = g supernatant of Leishmania and bovine PKII. The picture depicts an autoradiogram obtained after SDS-PAGE following photoaffinity incorporation under standard conditions of 1 ␮M 8-N3-w 32 PxcAMP in the absence and presence of 100 ␮M cAMP, respectively, to the samples indicated below. Lane 1, 50 ␮g of PKII; lane 2, 50 ␮g PKII q100 ␮M cAMP; lane 3, 100 000 = g supernatant from Leishmania; and lane 4, 100 ␮g of 100 000 = g supernatant from Leishmania q100 ␮M cAMP.

ŽBanerjee and Sarkar, unpublished observation.. It has been reported earlier that cAMP affects growth and differentiation in Leishmania, and components of the cAMP signaling pathway have been isolated. Unlike in higher eukaryotic system and unicellular eukaryotes such as T. cruzi, cyclic nucleotides seem to act in Leishmania promastigotes by a mechanism that does not involve any protein kinase. Although the catalytic subunit of PKA has been identified and purified and a gene c-lpk2 for the catalytic subunit of PKA have been obtained from Leishmania spp., several biochemical approaches have failed to detect the presence of any regulatory subunit of PKA in Leishmania dono¨ ani. The conceptual importance of this study in Leishmania is immense. This is the first report in eukaryotic systems, including Trypanosomatidae, of the absence of conventional regulation of the catalytic subunit of PKA via cAMP and regula-

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tory subunits. Temperature- and time-dependent degradation of c-lpk2 mRNA has led Simon-Tov et al. Ž1996. to the presumption of temperaturesensitive and post-transcriptional regulation rather than regulation via changes in cAMP. Further purification and elucidation of the function of these binding proteins are underway. In particular, it would be interesting to find a derivative of cAMP capable of binding selectively to one or more of the binding proteins and with poor affinity for the vertebrate counterparts or regulatory subunit of PKA. Such an approach could provide opportunities for rational drug design by manipulating the ability of these parasites to proliferate in the host environment.

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