Release of Ecto-protein Kinases by the Protozoan ParasiteLeishmania major*

Leishmania major promastigotes have externally oriented ecto-protein kinases (PK) that are capable of phosphorylating both endogenous membrane substrates and foreign proteins. Live parasites phosphorylate protamine sulfate, casein, and phosvitin but not bovine serum albumin. Addition of exogenous PK substrates, such as phosvitin or casein, induced the shedding of ecto-PK that are capable of phosphorylating protamine sulfate. No phosphorylation of protamine sulfate was seen when cell-free supernatants from promastigotes incubated with either buffer alone or bovine serum albumin were used. A second enzyme, a constitutively released PK that phosphorylates casein or phosvitin and not protamine sulfate or mixed histones, was identified and characterized. This PK is inhibited by 5 μm staurosporine, 50 μg/ml heparin, and 75 μm CKI-7, concentrations typical of the IC50 found for other eukaryotic casein kinases (CK). The constitutively shed ecto-PK specifically phosphorylated a peptide substrate for CK1 but not for CK2, suggesting that this shed PK is similar to CK1.

The protozoan parasite Leishmania is responsible for a wide spectrum of human diseases that cause varying degrees of patient morbidity and mortality and affect more than 12 million people world-wide. Leishmania have a relatively simple life cycle, existing as extracellular flagellated promastigotes in the sandfly vector and following transmission to a mammalian host, as intracellular aflagellated amastigotes in macrophages (1).
Throughout its life cycle, Leishmania encounter hostile, changing environments that require rapid responses to ensure survival of the parasites (1,2). In eukaryotes, protein phosphorylation is a major mechanism for regulating cellular responses to environmental signals, including cell-cell interactions. The role of intracellular protein kinases (PK, EC 2.7.1.37) 1 in complex regulatory cascades that control differentiation, metabolism, growth, gene expression, and other cellular processes is well established (3).
Less is known about the functions of externally oriented cell surface PK (ecto-PK), although the potential for their involvement in signal transduction and cell-cell interactions appears great. These ecto-PK utilize extracellular ATP that is present in blood plasma and other body fluids at concentrations from 1 to 30 M (4). Ecto-enzymes have been demonstrated in a variety of cultured cells, including HeLa cells, fibroblasts, neutrophils, neurons, and others (5)(6)(7)(8)(9)(10). Several types of serine/threonine ecto-PK have been identified and include the cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and cyclic nucleotide independent PK (6 -8, 10). Recently tyrosine ecto-PK have also been reported (11). Ecto-PK are capable of phosphorylating both endogenous membrane and exogenous foreign substrates. We have identified a cyclic nucleotide-independent ecto-PK activity on viable Leishmania major promastigotes that phosphorylate exogenous substrates, such as mixed histones and protamine sulfate, in addition to 11 endogenous parasite membrane proteins (12). Live parasites can also phosphorylate the C3 and C3b polypeptide components of the human complement system (13). Phosphorylation of C3 was shown to inactivate both the alternative and classical complement pathways (14) and thus may play an important role in parasite survival. In addition, the inducible release or shedding of ecto-PK in the presence of enzyme substrates has been described for specific PK on the surface of HeLa cells, endothelial cells, fibroblasts, neutrophils, and other cells (6,7,9,15,16). This activity appears to be similar to casein kinases and was recently purified from HeLa cells and characterized as casein kinase 1 (CK1) and casein kinase 2 (CK2, see Ref. 6).
In this study we show for the first time that parasites are capable of shedding ecto-PK. At least two leishmanial ecto-PK released by promastigotes were identified as follows: first, an ecto-PK that is shed constitutively and phosphorylates phosvitin; and second, an enzyme released by incubation with PK substrates that phosphorylates protamine sulfate. The constitutively shed enzyme was characterized and shown to be CK1like. These findings will allow us to further characterize the properties and roles of ecto-PK and the possible ramifications of these enzymes on host-parasite interactions.

EXPERIMENTAL PROCEDURES
Materials-The CK-specific inhibitors, CKI-7 and CKI-8, were purchased from Seikagaku America (St. Petersburg, FL). The CK1-and CK2-specific peptides, RRKDLHDDEEDEAMSITA and RRRADDSD-DDDD, respectively, used in the phosphorylation assays were generous gifts from Dr. L. Pinna (University of Padova, Italy). All the other PK inhibitors, protein substrates, and reagents were purchased from Sigma. P-81 phosphocellulose paper was obtained from Whatman Scientific Ltd. ( (17). Virulent cloned parasites were maintained by serial passage in BALB/c mice and obtained as required by needle aspiration from lesions. Parasites were maintained in culture for not more than 12 passages.
Release of Ecto-PK Activity-Viable promastigotes were washed once by centrifugation (10 min, 600 ϫ g) with 20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 1 mM MgCl 2 , 1 mM glucose, and 10 mM NaF (buffer A). The parasites were resuspended at 5 ϫ 10 8 cells/ml in buffer A (100 l) with or without substrate (1 mg/ml phosvitin, hydrolyzed or intact casein, or bovine serum albumin). The cells were layered over di-N-butyl phthalate (150 l) and incubated for 20 min at 30°C. The promastigotes were removed by rapid centrifugation in a Microfuge (Beckman model B, for 1 min), and the supernatants were used for phosphorylation assays.
Phosphorylation Assays-Released ecto-PK activity was measured in cell-free supernatants, prepared as described above, by adding PK substrates, [␥-32 P]ATP (1-10 Ci) and 0.1 mM cold ATP. The reactions were incubated for 10 -20 min at 30°C and stopped by the addition of ice-cold trichloroacetic acid (1 ml, 25%). After 30 min on ice protein phosphorylation was analyzed by either filtration or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the former case, the trichloroacetic acid precipitates were collected on Millipore HAWP filters (0.45 m), washed four times with ice-cold 5% trichloroacetic acid (2 ml each), and counted in a ␤-scintillation counter. For gel electrophoresis, the precipitate was collected by centrifugation and washed with 5% trichloroacetic acid (once) and 90% acetone (three times). The pellet was resuspended in sample buffer, analyzed by 12% SDS-PAGE, and exposed to x-ray film or phosphorimaging. Quantitation of the phosphorylation was carried out by densitometric analysis of the bands. The protein substrates and concentrations used are indicated in the figure legends.
Phosphorylation by live promastigotes. Parasites were washed once by centrifugation with buffer A and resuspended at 5 ϫ 10 8 cells/ml in buffer A (100 l). Cells were preincubated at 30°C with or without the PK substrates for 2-5 min, and the reaction was initiated by addition of [␥-32 P]ATP (1-10 Ci) and 0.1 mM cold ATP. After 10 -20 min incubation the reactions were stopped by adding trichloroacetic acid and analyzed by filtration and SDS-PAGE as described above.
Peptide Phosphorylation-Phosphorylation of specific peptide substrates for CK1 or CK2 using cell-free supernatants was carried out as described above with the following modifications. The reaction was stopped by the addition of ice-cold 100% trichloroacetic acid (24 l) and 1% bovine serum albumin (BSA, 40 l), and co-precipitated on ice (30 min). After centrifugation, samples from the supernatant (20 l in triplicate) were spotted on P-81 ion exchange chromatography phosphocellulose paper (Whatman Scientific Ltd., UK) and washed three times in 75 mM phosphoric acid (500 ml, 5 min each wash) to remove the unbound phosphate. The paper was dried and 32 P incorporation was measured in a ␤-scintillation counter.
Parasite Viability-Promastigote viability was assessed by two complementary assays as follows: ethidium bromide (EtBr) incorporation, which measures the percentage of dead or damaged cells; and fluorescein diacetate hydrolysis, which measures the percentage of viable cells. Fluorescein diacetate hydrolysis (18) was tested in selected experiments; however, the EtBr assay (19), described below, was used in all experiments. To determine the effect of each treatment on cell viability, promastigotes (5 ϫ 10 7 cells/200 l buffer A) were incubated in parallel under identical conditions, except for [␥-32 P]ATP, to those described above. At the beginning and end of each incubation 2.3 ml of buffer A containing 50 M EtBr was added, and the fluorescence was measured 5 min later in a fluorescent spectrophotometer (Perkin-Elmer LS-5B Luminescence Spectrometer, 365 nm excitation, 580 nm emission). Buffer A containing 50 M EtBr served as the blank. A standard curve using increasing numbers of promastigotes (5-500 ϫ 10 5 ) in buffer A containing digitonin (30 g/ml) and EtBr (50 M) was used to calculate the number of dead parasites. In some cases, the percentage of dead or damaged parasites was also determined by counting fluorescent and total parasites using a fluorescent phase microscope (Laborlux K; Leitz, Germany) at 400 ϫ magnification.
Effect of PK Inhibitors on Enzyme Activity-Stock solutions of the PK inhibitors heparin, CKI-7, CKI-8, staurosporine, H-7, and W-7 were prepared in dimethyl sulfoxide or distilled water. The inhibitors or dimethyl sulfoxide alone were diluted with buffer A and added to the reactions just before use. Phosphorylation of the PK substrates and analysis of the reactions by either filtration or SDS-PAGE was carried out as described previously. The final concentrations of inhibitors examined are given in the text.

Phosphorylation of Exogenous Protein Substrates by Ecto-
PKs on Live Promastigotes-The phosphorylation of different PK substrates or BSA was examined using viable L. major stationary phase promastigotes. After a 20-min incubation with [␥-32 P]ATP substrate, phosphorylation was analyzed by SDS-PAGE and autoradiography ( Fig. 1 and results not shown).
Leishmanial ecto-PK(s) are capable of phosphorylating several different exogenous substrates, including protamine sulfate (PS), phosvitin, hydrolyzed casein (h-casein), intact casein (i-casein), and mixed histones ( Fig. 1, data not shown and Ref. 12). Different phosphorylation patterns were observed for each PK substrate used. When phosvitin (lane 2) was added to the reaction mixture, one major radiolabeled band, 35 kDa, corresponding to the substrate molecular mass was observed. Likewise, when PS was added to the reaction mixture a major phosphorylated band at about 14 kDa, corresponding to this protein (lane 1), was seen. Phosphorylation of h-or i-casein gave as expected multiple bands or a major band at 29 kDa, respectively (lane 4 and data not shown). When BSA was used as an exogenous substrate, no protein phosphorylation was observed (lane 5). The only radioactive product observed using BSA was a band that migrated with the tracking dye. This band appears in all the reactions, including those without substrate (lane 3), and may represent phosphorylation of parasite lipids or small peptides. Endogenous phosphorylation of parasite proteins was only observed in the absence of foreign substrates and following extensive exposure of the gels (data not shown and Ref. 12). The additional high molecular weight bands observed when PS was used were not investigated but are probably due to protein impurities, since these bands were not observed in preliminary experiments when PS from an alternative commercial source was used (Ref. 12 and data not shown).
Parasite viability was measured by both EtBr fluorescence and fluorescein diacetate hydrolysis. In the absence of substrates, promastigote viability after 15 min at 30°C was never less than 97%, as measured by both methods, and remained essentially unchanged up to 35 min incubation. Incubation of the promastigotes with either PS, h-casein, or phosvitin for 25-35 min resulted in an 11, 2.5, and 0%, respectively, decrease in parasite viability compared with those incubated in buffer alone. Effects on parasite viability were already seen by 5 min incubation with the substrate, and essentially no additional change in viability (Ͻ1%) was noted with increasing time using any of the PK substrates. Similar differences in PK substrate toxicity for cells have been reported with HeLa cells, neutro- phils, and fibroblasts (5,7,8).
Release of Ecto-PK Activity from Parasites-Incubation of neutrophils, HeLa cells, and fibroblasts with substrates for PK was shown to induce the release of PK activity from these cells. This activity could be detected in the cell-free supernatants (7,11,15,16). Therefore, we decided to test if phosphorylation of exogenous substrates observed using live parasites was due to enzyme release by the cells. Promastigotes were washed and incubated with or without h-casein for 20 min. After removal of the cells by centrifugation through an oil layer, PK activity of the supernatants was assayed by adding PS where indicated ( Fig. 2; lanes 1-5).
Preincubation of promastigotes with h-casein resulted in the release of a PK that phosphorylates both h-casein and PS (lanes 1 and 4). Shedding of the PS phosphorylating activity by the cells required parasite preincubation with h-casein, since no phosphorylation of PS was observed when the parasites were preincubated in buffer alone (lane 2). Preincubation of promastigotes with other PK substrates, including i-casein, phosvitin, or PS, also caused the shedding of a PK activity that could phosphorylate PS. However, no phosphorylation of PS was observed if supernatants from parasites incubated with BSA were used (data not shown). The labeled bands seen in lanes 1 and 4 are not due to phosphorylation of endogenous secreted parasite proteins, since no radiolabeled bands were observed if supernatant alone was used in the phosphorylation reactions (lane 5). Likewise, PS was not phosphorylated when added to the reaction mixture in the absence of supernatants collected from h-casein-treated promastigotes (lane 3).
Shedding of the PK that phosphorylates PS requires promastigote preincubation with h-casein. However, it was not clear whether h-casein phosphorylation (Fig. 2, lane 1) required parasite preincubation with substrate, "induced release," or if this PK activity was constitutively released by the parasites. Cellfree supernatants were collected from promastigotes incubated with or without h-casein (Fig. 2, lanes 6 and 7). Substrate was added to supernatant obtained from parasites incubated in buffer alone and the phosphorylation reaction carried out. Phosphorylation of h-casein was seen when either procedure was used and did not depend on whether the parasites were incubated with the substrate prior to collection of the supernatant. These results demonstrate that the PK activity that phos-phorylates h-casein is constitutively released from the leishmanial parasites. However, phosphorylation of h-casein using supernatants collected from parasites preincubated with substrate was 70% greater than those only incubated with buffer ( lanes 6 and 7), suggesting that incubation with casein may induce release of a PK activity capable of phosphorylating h-casein. These findings suggest that at least two different kinase activities are released from intact cells: 1) "substrateinducible" activity that phosphorylates PS and perhaps h-casein; and 2) a "constitutive" activity that only phosphorylates h-casein and not PS. The "constitutively" shed leishmanial CK-like activity, LCK, was characterized further.
Substrate Specificity of the Constitutive PK Activity-Enzyme activity of cell-free supernatants, collected from promastigotes incubated with buffer alone, was measured using several different PK substrates, including h-casein, i-casein, phosvitin, mixed histones, PS, and BSA. Phosphorylation was examined by SDS-PAGE ( Fig. 3 and data not shown). Phosvitin was the best substrate for the constitutively released PK (lane Effect of Inhibitors on the Constitutively Released PK-Several different PK inhibitors, including staurosporine, W-7, heparin, CKI-7 and CKI-8, were examined for their ability to block phosvitin phosphorylation. The antibiotic staurosporine, a competitive inhibitor of ATP binding to PK, inhibits a wide range of enzymes, including PKC, PKA, and Ca 2ϩ -calmodulin PK, at nanomolar concentrations. Unlike most PK, CK are less sensitive to staurosporine, and the IC 50 for CK1 and CK2 is 163 and 19 M, respectively (20). The IC 50 found for the leishmanial CK (LCK), 5 M, is similar to CK2.
Heparin has been used to distinguish between the two CKs (21). CK2 is strongly inhibited at approximately 1% the concentration that inhibits CK1 (IC 50 Ϫ CK1 ϭ 24 g/ml; CK2 ϭ 0.15 g/ml). Phosphorylation of phosvitin by LCK was measured in the presence of different concentrations of heparin (Fig.  4). This curve shows that relatively high concentrations of heparin (IC 50 ϭ 50 g/ml), more similar to CK1, are needed to inhibit the leishmanial enzyme's activity. In addition to heparin, the effect of CKI-7, an isoquinoline derivative of W-7, on phosvitin phosphorylation was also examined. CKI-7 is a specific inhibitor of CK1 and CK2 (IC 50 Ϫ CK1 ϭ 9.5 M; CK2 ϭ 90 M; see Ref. 22). The IC 50 for other common PK, such as PKA, PKC, and Ca 2ϩ -calmodulin PK, is much higher, 550, Ͼ1000, and 195 M, than for CK. The IC 50 found for LCK was 75 M, closer to that observed for mammalian CK2. Taken together the results using PK inhibitors and substrate specificity strongly suggests that the shed leishmanial enzyme is a CK. LCK was also inhibited by W-7 (IC 50 Ϫ 10 M).
Phosphorylation of CK1-and CK2-specific Peptide Substrates-The constitutively released LCK activity and total cellular CK activity were further characterized in four separate experiments using peptides specific for either CK1 (RRKDLH-DDEEDEAMSITA) or CK2 (RRRADDSDDDDD; Ref. 23). Results typical of these experiments are give in Table I. Phosphorylation of the peptides using parasite lysates, either freeze/ thawed or sonicated, showed that both enzymes are present in the parasite (Table I and data not shown). The total activity found in the promastigote lysates for CK1 was approximately 2.6-fold higher than CK2.
When peptide phosphorylation was examined using cell-free supernatants as a source of LCK, the activity was significantly lower than that measured using parasite lysates. In some experiments both CK-specific peptides were phosphorylated (data not shown). However, the CK1 activity was consistently higher, up to 650 times greater, than the CK2 activity (Table I). Phosphorylation of the CK-specific peptides using intact promastigotes gave results similar to that found using the cell-free supernatants. These results strongly suggest that the constitutively released leishmanial enzyme is CK1-like. Furthermore, the finding of high CK1 activity in the cell-free supernatant (9.6% of the parasite lysate) and little or no CK2 activity suggests that the released activity is not due to cell lysis. Promastigote viability in buffer A was Ͼ97% over 20 min.
Kinetics of Constitutive PK Release-Release of the CK-like activity over time was followed for 30 min. At each time point, aliquots containing parasites in buffer alone were removed and cell-free supernatants prepared. In parallel, the percentage of dead promastigotes was measured using the EtBr assay. CK-like activity was assayed using phosvitin as substrate and analyzed by SDS-PAGE and densitometry. Results from one typical experiment is shown in Fig. 5. PK activity shed by the parasites into the buffer increased dramatically over the first 10 min of incubation (600%). After peaking, the activity measured in the cell-free supernatants slowly decreased, until it appeared to level off after 25 min at twice the initial activity. The initial time point (t ϭ 0 min) was obtained by adding parasites to buffer and then immediately centrifuging to prepare a cell-free supernatant. All time points were compared with a negative control, phosvitin, in labeling buffer without supernatant. The percentage of dead promastigotes was examined in parallel by EtBr staining and showed no change in parasite viability over the first 15 min in labeling buffer (98% viable; t ϭ 0 and 15 min) and only a small decrease after 30 min (96% viable). The difference in kinetics of CK secretion and change in parasite viability further excludes the possibility that the PK release observed is a result of cell damage. DISCUSSION Many eukaryotic cells possess ecto-PK that are capable of phosphorylating foreign and endogenous protein substrates. PK identified include PKC, PKA, CK1, and CK2. In addition, several vertebrate cells were shown to shed ecto-PK from their surface in the presence of PK substrates like phosvitin or casein. Although most cells examined appear to release cyclic nucleotide independent CK, the release of PKA or PKC has been documented in only a few cases.
Parasites have evolved varied strategies to evade host defense mechanisms including the mimicry of host regulatory molecules and enzymes. Leishmania promastigotes also express ecto-PK on their surface that phosphorylate foreign proteins (24). Previous studies showed little or no evidence that the parasite ecto-PK activity was related to PKA or PKC, respectively. Activators and inhibitors of these enzymes had no significant effect on the phosphorylation of exogenous or endogenous substrates (Ref. 12 and data not shown). However, comparative studies between live parasites, which phosphorylate the C3 and C3b polypeptides of the human complement system, and LPK-1, a purified parasite enzyme, which only phosphorylates C3, but not C3b, suggested that promastigotes possess more than one ecto-PK (13). Preliminary experiments show that the constitutively shed leishmanial PK, LCK, phosphorylates C3a (data not shown). Interestingly, a casein kinase shed from human platelets following activation was shown to FIG. 4. Effect of heparin on the constitutively shed leishmanial casein kinase (LCK) activity. Promastigotes were incubated in labeling buffer for 20 min at 30°C, and the cell-free supernatants were collected by centrifugation through an oil layer. Protein kinase activity was assayed using phosvitin (1 mg/ml), [␥-32 P]ATP, and increasing concentrations of heparin. The reaction was stopped after 10 min by the addition of ice-cold trichloroacetic acid and analyzed by 12% SDS-PAGE and autoradiography. Quantitation of the phosvitin phosphorylation was carried out by densitometry.

TABLE I
Phosphorylation of casein kinase (CK) peptide substrates by leishmanial protein kinases Shed cLPK, promastigotes (5 ϫ 10 7 /100 l) were incubated for 20 min at 30°C and removed by centrifuging through an oil layer. The cell-free supernatants were used as a source of enzyme. Lysed parasites, promastigotes (5 ϫ 10 7 /100 l) were freeze/thawed three times and used as a source of enzyme. Intact parasites, live promastigotes (5 ϫ 10 7 /100 l) were incubated directly with the CK-specific peptide substrates. CK1 (RRKDLHDDEEDEAMSITA) or CK2 (RRRADDSDDDDD) specific peptide, [␥ 32 P]ATP, and protein kinase were incubated for 10 min, and the reaction was stopped by adding 1% BSA and trichloroacetic acid. After 30 min on ice the precipitate was removed by centrifugation, supernatants in triplicate were spotted on P-81 filters and washed with 75 mM phosphoric acid, and 32 P incorporation was measured by liquid scintillation counting. Background  phosphorylate both C3 and C3b (25). To obtain a better understanding of the role of ecto-PK in host-parasite interactions, it will be necessary to characterize parasite ecto-enzymes and the physiological substrates involved in these processes.
In this study we demonstrate that Leishmania promastigotes have at least two types of ecto-PK, both of which can be released by the parasites. Unlike most cells examined so far, one type of activity appears to be shed continuously in the absence of substrate, whereas the second activity, similar to other eukaryotic cells, is shed only when incubated with substrate. These activities, constitutive and induced, are easily distinguished by their ability to phosphorylate PS, since the former enzyme(s) shows no activity when assayed with this substrate, and the latter readily phosphorylates PS.
These shed ecto-PK are not due to cytoplasmic leakage from damaged or dead cells. Although both histone and PS are cytotoxic for eukaryotic cells (5,16,26) and parasites, little or no cytotoxicity was found when cells (5,7,16,26) or parasites were incubated with phosvitin or casein. Initial parasite viability in the studies described herein always exceeded 97%, as measured by two fluorescent assays, and changed by Ͻ2% following incubation either in the presence or absence of casein or phosvitin. This percentage was identical to that found following incubation with buffer alone. The small amount of dead parasites was not responsible for the induced PK activity observed, since no phosphorylation of PS was noted using cell-free supernatants from promastigotes incubated either with BSA or buffer alone, whereas good phosphorylation of PS was found with as little as 2.5 ϫ 10 6 freeze/thawed parasites (data not shown). Furthermore, no correlation was found between the release of constitutive ecto-PK activity in the cell-free supernatants and decreasing cell viability. Ecto-PK activity peaked rapidly at 10 min and then gradually decreased with time, whereas cell viability remained essentially constant over the first 15 min and then decreased only slightly, by 2%, after 30 min.
We decided to focus on the characterization of the constitutively shed leishmanial ecto-PK activity that appears to be related to casein kinases. Substrate specificity was typical of these enzymes. Phosvitin, followed by h-casein and i-casein, was the best substrate for the enzyme(s), and neither mixed histones nor PS were phosphorylated by the ecto-PK.
The parasite activity is different from a spontaneously shed human leukemic cell line serine/threonine ecto-PK that was recently reported (11). The latter activity phosphorylates PS and histone H2B, as well as casein, phosvitin, and the human complement polypeptide C9, and may contain more than one PK. PKI, a specific PKA inhibitor, was found to inhibit the phosphorylation of histone H2B but not C9 by the leukemic cell ecto-PK. The latter activity was postulated to be CK-like. Little evidence was found in our study or previous studies for either an intracellular or externally oriented leishmanial PKA (12). However, we have recently cloned and characterized the genes for two PKA catalytic subunits from L. major (27).
The effect of several PK inhibitors on the leishmanial ecto-PK was examined. Unlike most PK that are inhibited by nanomolar concentrations of staurosporine (20), the IC 50 values for casein kinases are in the M range (CK1 and CK2, 163 and 19 M, respectively). The IC 50 value for the leishmanial ecto-PK (5 M) was similar to CK2. The high concentration of drug need to inhibit the leishmanial activity is not due to the intrinsic resistance of parasite enzymes to staurosporine. The phosphorylation of PS by Leishmania aethiopica promastigotes, either particulate or soluble fractions or live parasites, is strongly inhibited by staurosporine. Low concentrations of drug (50 nM) inhibited the phosphorylation of PS by Ͼ80% when parasite fractions were used and by approximately 45% using live parasites (28). Furthermore, staurosporine concentrations similar to those that inhibit the constitutively shed ecto-PK are cytostatic and/or cytotoxic to the promastigotes and induce pronounced morphological changes (29).
Heparin and CKI-7, both specific CK inhibitors, also blocked the leishmanial ecto-PK activity at concentrations similar to those reported for mammalian and yeast CK and confirmed that the constitutively shed parasite enzyme is CK-like. However, no conclusion regarding the type of CK in the cell-free supernatants could be made based on IC 50 values for these inhibitors, since the constants found using heparin or CKI-7 each implicated the presence of a different CK, either CK1 or CK2, respectively. Interestingly, heparin concentrations (10 M) similar to those that inhibit LCK were also shown to significantly reduce endogenous protein phosphorylation by live parasites (30).
Finally, we were able to identify the constitutively shed PK by examining the phosphorylation of specific peptide substrates for CK1 and CK2. Only the CK1-specific peptide substrate was phosphorylated confirming that the constitutively shed LCK activity is CK1-like. This conclusion was further supported by the finding that the LCK, similar to other CK1, only utilizes ATP, whereas CK2 utilizes both ATP and GTP for phosphorylation (data not shown).
Although spontaneously shed ecto-PK activity has not been observed with cell-free supernatants from HeLa cells or neutrophils (7,16), ecto-CK released from HeLa cells by incubation with phosvitin were recently purified and characterized (6). Differences in the sensitivity of the leishmanial and mammalian ecto-PK to different inhibitors suggest that it may be possible to design drugs that specifically inhibit the parasite but not the host enzymes. However, this will require the purification and characterization of the parasite enzyme(s).
The CK1 family has been found in all eukaryotic cells exam- FIG. 5. Constitutive secretion of leishmanial casein kinase 1 (LCK1) by promastigotes of L. major. Effect of incubation time on released protein kinase activity and cell viability was measured. Promastigotes in labeling buffer were layered on oil and incubated at 30°C. At three time points samples were taken to check parasite viability by staining with ethidium bromide and counting in a fluorescent microscope. In parallel, the promastigotes were removed by rapid centrifugation, and the cell-free supernatants were assayed for LCK1 activity by the addition of phosvitin and [␥-32 P]ATP. Reactions were analyzed by 12% SDS-PAGE, autoradiography, and densitometric scanning of the film. F, LCK1 activity; f, parasite viability. ined so far and is believed to be involved in the regulation of nuclear and cytoplasmic processes. These PK consist of monomeric proteins that vary considerably in size from 25 to 55 kDa and have been found in the nucleus, cytoplasm, membrane, and cytoskeleton. Several different isoforms of CK1 have been identified in mammalian cells and yeast using molecular techniques. In Saccharomyces cerevisiae two essential genes have been sequenced and found to encode a carboxyl-terminal prenylation motif, believed to target them to the plasma membrane. Using a nested polymerase chain reaction with degenerate oligonucleotides to conserved regions of CK1, we have amplified a 342-base pair fragment from L. major that shows 74.4% identity over 336 base pairs to human CK1-⑀. 2 We expect that molecular analysis of the leishmanial-CK1 gene (lck1), the recent availability of molecular techniques for the production of null Leishmania mutants, and further biochemical characterization of parasite ecto-PK will prove invaluable in understanding the role of these enzymes in parasite-host interactions.