Eukaryotic Cell
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cuevas, I. C.
Right arrow Articles by Docampo, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cuevas, I. C.
Right arrow Articles by Docampo, R.

 Previous Article  |  Next Article 

Eukaryotic Cell, September 2005, p. 1550-1561, Vol. 4, No. 9
1535-9778/05/$08.00+0     doi:10.1128/EC.4.9.1550-1561.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Characterization of Farnesylated Protein Tyrosine Phosphatase TcPRL-1 from Trypanosoma cruzi{dagger}

Ileana C. Cuevas,1,2 Peter Rohloff,2 Daniel O. Sánchez,1* and Roberto Docampo2,3*

Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, Universidad Nacional de General San Martín, Consejo Nacional de Investigaciones Científicas y Técnicas, San Martín, Provincia de Buenos Aires 1650, Argentina,1 Laboratory of Molecular Parasitology, Department of Pathobiology and Center for Zoonoses Research, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802,2 Department of Cellular Biology and Center for Tropical and Global Emerging Diseases, The University of Georgia, Athens, Georgia 306023

Received 11 March 2005/ Accepted 6 April 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Protein tyrosine kinases and phosphatases play important roles in the regulation of cell growth, development, and differentiation. We report here the identification in Trypanosoma cruzi of a gene (TcPRL-1) encoding a protein tyrosine phosphatase. The predicted protein (TcPRL-1) shares ca. 35% identity with the mammalian protein tyrosine phosphatase known as phosphatase of regenerating liver 1 (PRL-1). Four copies of this protein tyrosine phosphatase are present in the T. cruzi genome, and Northern blot assays showed a transcript of ~750 bases. TcPRL-1 was detected by Western blot analysis only in amastigote extracts as a 21-kDa protein. TcPRL-1 was expressed in Escherichia coli, and its phosphatase activity was determined by using p-nitrophenylphosphate and a phosphorylated protein as substrates. In contrast to other PRLs, TcPRL-1 activity was not affected by pentamidine, and it was inhibited by very low concentrations of o-vanadate. TcPRL-1 has a C-terminal CAAX motif (CAVM) and is farnesylated in vitro by T. cruzi epimastigote extracts and in vivo according to the transfection results. After transfection of T. cruzi with a vector that expresses TcPRL-1 as a C-terminal fusion to green fluorescent protein, GFP-TcPRL-1 was detected in the endocytic pathway of epimastigotes, amastigotes, and trypomastigotes by colocalization with cruzipain and concanavalin A. Interestingly, a mutant form without the CAAX motif localized to the cytoplasm, in contrast to its mammalian counterparts that localize to the nucleus. The results of these studies on TcPRL-1 reveal that, even though the animal and parasite PRLs share similar kinetic properties, their susceptibilities to inhibitors, as well as their localization, are distinct, implying that they may be involved in different cellular processes.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Phosphorylation of proteins in specific tyrosyl residues is a major control mechanism for several processes such as normal cell growth, differentiation, metabolism, cell cycle, cell migration, and gene expression, among others (16). The levels of cellular protein phosphorylation in tyrosine residues are controlled by the activities of both protein tyrosine kinases and phosphatases. Although protein tyrosine phosphatases (PTPs) were initially believed to have housekeeping roles, it is now obvious that they are highly regulated and specific enzymes (33).

PTPs form a large superfamily of enzymes with more than 100 members. The hallmark that defines the PTP superfamily is the presence of the signature motif (or active site), HCX2GX2R in the catalytic domain. PTPs can be divided according to their sequence homology and substrate specificity in tyrosine-specific phosphatases and dual-specific phosphatases. The latter class of phosphatases are the only that cleave phosphoester bonds in proteins that contain phosphotyrosine (pTyr), as well as phosphoserine and phosphothreonine.

The phosphatase of regenerating liver (PRL) phosphatases represents a new class of PTP. These phosphatases, in mammals, belong to a family composed by at least three members (PRL-1, -2, and -3) with an identity of >70% among them (38). It is interesting that in addition to the PTP signature motif they show homology, to a lesser extent, to cdc14p and PTEN/MMAC1, both dual specificity phosphatases (38). PRL phosphatases are small enzymes with an apparent molecular mass of 20 to 22 kDa.

An interesting feature of PRL phosphatases is the presence of a C-terminal CAAX motif as the signal for protein prenylation, where C is a cysteine, A is an aliphatic amino acid, and X is any amino acid. A protein farnesyltransferase (PFT) transfers farnesyl pyrophosphate (FPP) to the CAAX motif when X is Met, Ser, or Gln, whereas protein geranylgeranyltransferase I (PGGT-I) prefers Leu in the X position. In in vitro assays, all PRL family members are substrates of PFT (5) and PRL-1 and -2 also of PGGT-I (39). Furthermore, they are also farnesylated in vivo (39).

PRL-1 is able to dephosphorylate pTyr substrates, including itself, in vitro (11). PRL-1 was originally found predominantly in the cell nucleus (11). In CHO cells, PRLs were found associated with the plasma membrane and early endosomes when farnesylated and translocated to the nucleus when unlipidated (39). Recently, endogenous PRL-1 was found in the endoplasmic reticulum of nonmitotic cells and associated with centrosomes and the mitotic spindle of mitotic cells (34).

PRL-1, the first PRL described, was identified as an immediate-early gene whose expression was induced in mitogen-stimulated cells and regenerating liver (11). In intestinal epithelia, PRL-1 expression is associated with cellular differentiation but not proliferation (12, 39). Further, PRL-1 mRNA expression is elevated in several tumor cell lines but low in proliferating, nontumorigenic cells (34). The correlation of PRL-1 expression with growth and/or differentiation suggests that PRL-1 may have different roles depending on the cell type. Regarding its function, several data indicate that PRL-1 may be implicated in the control of cell growth, possibly by modulating spindle dynamics or proper spindle function (34) or by stimulating progression from G1 to S phase (35).

The protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease, is an important human pathogen with a digenetic life cycle involving insect and vertebrate hosts. During its complex life cycle, the parasite undergoes morphological and physiological changes. These cellular events, such as cell division, differentiation, and host cell invasion, could involve kinases, as well as phosphatases. Little is known about tyrosine phosphatases in trypanosomatids, although previous studies have reported the presence of PTP activities in T. cruzi (1, 15), Trypanosoma brucei (1, 2), and Leishmania donovani (6). We therefore searched for molecular evidence for the presence of these enzymes in T. cruzi.

PRLs have been found in several animal species (5, 11, 36, 39). However, proteins belonging to this family have not yet been described in unicellular eukaryotes. We report here the isolation, expression, characterization, and subcellular localization of a PRL phosphatase from T. cruzi. The protein is farnesylated in vitro and in vivo, and farnesylation is necessary for its novel localization in the endocytic pathway of T. cruzi.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures. T. cruzi CL Brener cloned stock (41) and, where indicated, the Y strain were used. Different forms of the parasites were obtained as previously described (14, 15). The purity of the different forms of the parasites (epimastigote, tissue culture-derived trypomastigote, and amastigote) was examined by conventional microscopy and was at least 95%.

Chemicals and reagents. Fetal bovine serum, Dulbecco phosphate-buffered saline (PBS), glutathione-Sepharose 4B column, protein A-Sepharose, poly-lysine, thrombin, [3H]FPP (15 Ci/mmol), p-nitrophenylphosphate (p-NPP), o-vanadate, okadaic acid, pentamidine [1,5-di(4-amidinophenoxy)pentane], myelin basic protein (MBP), recombinant human Src kinase, and protease inhibitors were purchased from Sigma Chemical Co. Restriction enzymes, TRIzol reagent, and Geneticin (G418) were from Gibco-BRL/Life Technologies, Inc. (Gaithersburg, MD). pGEM-T-Easy vector was from Promega. [{alpha}-32P]dCTP (3,000 Ci/mmol), pGEX2T, and the enhanced chemiluminescence detection kit were obtained from Amersham Biosciences. Alexa 488- and Alexa 546-labeled antibodies were from Molecular Probes, Inc. (Eugene, OR). Zeta-Probe nylon membranes were from Bio-Rad. Keyhole limpet hemocyanin was from Pierce. [{gamma}-32P]ATP (5,000 Ci/mmol) was from New England Nuclear Life Science Products (Boston, MA). Monoclonal antibody 212-BH6 against cruzipain (22) was a gift from Julio Scharfstein (Federal University of Rio de Janeiro, Rio de Janeiro, Brazil). All other reagents were analytical grade.

Gene cloning and expression. For mapping the TcPRL-1, we carried out mini-exon-heminested reverse transcription-PCR against T. cruzi total RNA (5 µg). The coding region of TcPRL-1 was retrieved by PCR on genomic T. cruzi DNA using a forward primer (TcPRL-1.N, 5'-ATG GGG GCC AAC GGC ACG-3') and a reverse primer (TcPRL-1.C, 5'-CTA CAT GAC CGC ACA CCC-3'). A mutant form of TcPRL-1 (TcPRL-1{Delta}CAVM) lacking the four C-terminal amino acids (173 to 176) of the CAAX domain was obtained. For this, TcPRL-1 was amplified with the same forward primer as described above in conjunction with a reverse primer corresponding to the sequence encoding amino acids 169 to 172 (5'-CTA CCC CGC GCA ACT-3'). Catalytically inactive TcPRL-1 (TcPRL-1C107S), in which the active site Cys residue (Cys107) was replaced by Ser, was obtained through PCR using forward and reverse primers corresponding to the desired nucleotide substitution (5'-GTG CAC TCC GTT GCT GGA-3', 5'-AGC AAC GGA GTG CAC GGC AAT-3'). The templates used were TcPRL-1 for TcPRL-1C107S and TcPRL-1{Delta}CAVM for TcPRL-1C107S{Delta}CAVM. The PCR product was then ligated and transformed in Escherichia coli. All PCR fragments were cloned in pGEM-T-Easy vector. For protein expression, wild-type, TcPRL-1{Delta}CAVM, and TcPRL-1C107S fragments were subcloned in pGEX2T. Radioactive probes were obtained by amplification of TcPRL-1 with TcPRL-1.N/TcPRL-1.C. Plasmid constructs were confirmed by sequencing using an ABI 377 DNA sequencer (Perkin-Elmer). All sequence computational analyses was done by using the Lasergene package from DNASTAR, Inc. The alignments were done by using on-line Workbench server from UCSD (http://workbench.sdsc.edu).

Southern and Northern blot analysis. DNA was prepared from epimastigotes of T. cruzi using a conventional proteinase K and phenol-chloroform method (28). DNA was digested with the indicated restriction enzymes. RNA was purified using TRIzol reagent according to the manufacturer's instructions. Southern and Northern blots were performed as described previously (28). DNA and RNA were transferred to Zeta-Probe nylon membranes and UV cross-linked. High specific radioactivity probes were obtained labeling with [{alpha}-32P]dCTP by PCR as described previously (20). Filters were hybridized with the probe described above, using a hybridization solution containing 7% sodium dodecyl sulfate (SDS), 1% bovine serum albumin, 3x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), and 1 mM EDTA and washed at 63°C in 0.2x SSC-0.1% SDS.

Expression of recombinant protein in bacteria. The different TcPRL-1 (wild-type, {Delta}CAVM, and C107S) were expressed as fusions with glutathione S-transferase (GST). After induction at 30°C to produce soluble protein, the different forms were purified according to the manufacturer instructions using a glutathione-Sepharose 4B column. Soluble purified TcPRL-1 was obtained by in column digestion overnight at 4°C with 50 U of thrombin prepared in PBS. After recovery and dialysis against 5 mM HEPES (pH 7.5), the purity of the preparation was checked by SDS-polyacrylamide gel electrophoresis (PAGE).

Parasite extracts. PBS-washed parasites were resuspended to 109 parasites/ml in PBS containing 1% Nonidet P-40 (NP-40), 1 mM phenylmethysulfonylfluoride, 0.5 mM TLCK (N{alpha}-p-tosyl-L-lysine chloromethyl ketone), 5 mM EDTA, and 10 mM N-(trans-epoxysuccinyl)-L-leucine-4-guanidinobutylamide (E-64). The suspension was kept on ice for 10 min with frequent mixing and centrifuged at 3,000 x g at 4°C for 10 min. The supernatant and the pellet, resuspended in the same buffer solution, were kept and used for the SDS-PAGE experiments.

PAGE and Western blot analysis. Polyacrylamide gels were prepared according to (28). Antibodies against TcPRL-1 were obtained by using the synthetic peptide PepTcPRL-1 (WLMRYKPRHQEGNEGSLSCC), corresponding to amino acid residues 152 to 170 of TcPRL-1. The C-terminal Cys was added for the covalent linking of the peptide to the carrier keyhole limpet hemocyanin. Rabbit polyclonal anti-TcPRL-1 serum prepared against PepTcPRL-1 was raised in the Immunological Resource Center, University of Illinois at Urbana-Champaign. Antibodies to the whole recombinant protein were obtained by immunization of a rabbit with GST-TcPRL-1. Western blot analysis and detection by enhanced chemiluminescence were performed as described previously (28). Wells were loaded by using lysates from equivalent number of cells or protein as stated.

In vitro prenylation assays. Prenylation reactions were done as described elsewhere (23) with some modifications. A T. cruzi epimastigote extract was used as a source of prenyltransferase. The extract was prepared by sonicating 1.2 x 108 parasites in a buffer containing 1 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 1 mM dithiothreitol (DTT). The extract was then supplemented with 20 mM Tris-HCl (pH 8.0), 5 mM DTT, and 5 µM ZnCl2. The lysate was clarified by centrifugation in a microcentrifuge at 12,000 rpm for 30 min at 4°C, and the supernatant was used in the prenylation reactions. The prenylation mixture contained 5 mM DTT, 0.5 mM MgCl2, 20 µM ZnCl2, 30 mM potassium phosphate (final pH 7.7), 2 µg of TcPRL-1 or TcPRL-1-{Delta}CAVM fused to GST as substrate, and 40 µg of epimastigote extract. A total of 2 µCi of [3H]FPP was used as isoprenoid donors. The reaction mixture (40-µl total volume) was incubated at 30°C for 3 h, boiled, and resolved by SDS-10% PAGE. The gel was incubated in En3Hance, dried, and exposed to film at –80°C for 1 week.

Phosphatase activity determination. For phosphatase assays ~10 µg of recombinant TcPRL-1 was used per reaction in a total volume of 50 µl. Assays were performed in a reaction mixture containing 50 mM HEPES (pH 7.5), 0.1% 2-mercaptoethanol, and 10 mM p-NPP. The mixture was incubated for 1 h at 37°C, and the reaction was stopped by the addition of 10 mM o-vanadate. The results were determined by using a spectrophotometer at 405 nm. Inhibition studies were done by adding increasing concentrations of sodium o-vanadate, okadaic acid, EDTA, NaF, or pentamidine from stock solutions to the activity assay. For the determination of substrate turnover, standard curves were established from serial dilutions of the corresponding unphosphorylated compound (p-NP). Reactions performed under the same conditions but in the presence of TcPRL-1C107S were used as controls for the absence of contamination from bacteria in the enzyme preparation. The results are expressed as means ± the standard deviation values of triplicate samples.

In vitro dephosphorylation of tyrosine-phosphorylated MBP by TcPRL-1. Wild-type and TcPRL-1C107S were purified by thrombin digestion as outlined above. Tyrosil residues of MBP were radioactively labeled with [{gamma}-32P]ATP and recombinant human Src kinase according to the manufacturer's instructions. After stopping the reaction, different amounts of TcPRL-1 were incubated with ~2 µg of pTyr-MBP at 37°C for 1 h in reaction buffer as described above. The phosphatase reaction was terminated by the addition of an equal volume of 2x Laemmli buffer. The samples were then boiled, separated by SDS-12% PAGE, and exposed to X-ray film. Densitometric analysis of the protein bands was done by using the program ImageJ (http://rsb.info.nih.gov/ij/).

GFP tagging and T. cruzi transfection. Wild-type and mutant forms of TcPRL-1 open reading frames (ORFs) were digested and subcloned into the T. cruzi expression vector pRIBOTEX (19) fused to the C terminus of the green fluorescent protein (GFP). A total of 50 µg of QUIAGEN purified DNA was used to transfect T. cruzi CL Brener as previously described (27). Stable transfectants were selected and expanded in 500 µg of G418/ml and screened by observation under a fluorescence microscope. To confirm the correct expression of fusion proteins, we performed immunoprecipitations of transfected parasites using anti-GFP serum and protein A-Sepharose. Transfections of Y strain epimastigotes were carried out in a 2-mm gap cuvette with a Bio-Rad Gene Pulser II set at 1.5 kV and 50 µF. Four x 107 parasites were harvested, washed twice with 10 ml of HBS buffer (21 mM HEPES [pH 7.5], 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM glucose) at 3,000 x g for 5 min, and resuspended in 0.4 ml of HBS with 50 µg of plasmid DNA (pRIBOTEX or pTcPRL-1/RIBOTEX). Parasites were recovered in 5 ml of liver infusion tryptose medium supplemented with 10% fetal bovine serum at 28°C and, after 24 in culture, G418 was added to a final concentration of 250 µg/ml. Parasites were cloned by limiting dilution in 96-well plates. Transfected epimastigotes were differentiated to mammalian forms as described previously (21). To assess the efficiency of amastigote to trypomastigote differentiation in TcPRL-transfected cells, monolayers of myoblasts in 25-cm2 tissue culture dishes were infected for 6 h with 4 x 106 trypomastigotes. The relative percentage of amastigote and trypomastigote forms was determined in culture supernatants 5 days postinfection.

Fluorescence microscopy. For cruzipain colocalization studies, parasites fixed with 4% paraformaldehyde were allowed to adhere to poly-L-lysine-coated coverslips; permeabilized with 0.3% Triton X-100 for 5 min; and blocked with 3% bovine serum albumin, 1% fish gelatin, 50 mM NH4Cl, and 5% goat serum in PBS for 1 h. The cells were stained with anti-cruzipain (1:200), followed by Alexa 546 goat anti-mouse antibody (1:1,000). For concanavalin A colocalization, live parasites were incubated for 30 min in a buffer containing 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 50 mM HEPES (pH 7.4), and 5 µg of concanavalin A-TRITC/ml. Cells were then fixed and adhered to coverslips as described above. Confocal images were collected with a Leica laser scanning confocal microscope (TCS SP2) by using a x63 Plan-Apo objective lens with NA 1.32. Single optical sections were recorded with an optimal pinhole of 0.000293 according to Leica instructions. Adobe Photoshop was used for image processing.

Nucleotide sequences. The nucleotide sequence reported here has been submitted to the GenBank Data Bank under accession number AY461711.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of T. cruzi PRL-1. Searching a database from our laboratory with rat PRL-1 amino acid sequence (GenBank NP_113767.1) led to the identification of a T. cruzi expressed sequence tag (EST) clone that could encode a protein homologous to PRL-1. We mapped the 5' untranslated region (5'UTR) of TcPRL-1 mRNA by RT-PCR, and by PCR we obtained the coding region of TcPRL-1 for T. cruzi PRL-1. Sequencing revealed an ORF of 531 bp coding for a 176-amino-acid protein with a predicted molecular mass of 20 kDa and an isoelectric point of 7.7.

Figure 1A shows the alignment of TcPRL-1 with the orthologues from T. brucei (AAX69332 [GenBank] .1), Homo sapiens (NP_003454 [GenBank] .1), Drosophila melanogaster (AAF53506 [GenBank] .1), and Caenorhabditis elegans (AAC17103 [GenBank] .2). Since human, mouse, and rat PRL-1s are 100% identical, we used the human sequence as representative of the mammalian orthologues in the alignment. All five proteins displayed in Fig. 1A show a 100% identity within the catalytic domain and a similar length. TcPRL-1 shows the consensus motif for PTP activity (boxed in Fig. 1A), with the Cys107 essential for activity conserved (Fig. 1A, asterisk). Other residues present in PRLs and conserved in TcPRL-1 include (i) the Asp75, which serves as a general acid in the catalysis; (ii) an Ala instead of a Ser or Thr next to the catalytic Arg113, which explains the low catalytic efficiency of PRLs compared to other PTPs (17); and (iii) the Cys52 that could form an intramolecular disulfide bond with the Cys107 and prevent its oxidative damage (17). Like all PRLs, TcPRL-1 also has the CAAX motif (underlined in Fig. 1A) in the C terminus. However, the T. cruzi CAAX motif is different from the ones present in other PRLs but similar to the prenylation motif present in the T. brucei PRL and to those described in other trypanosomatids (37). As shown in Fig. 1B, the percentage of identity between TcPRL-1 and other PRLs ranges from 34 to 50%.



View larger version (60K):
[in this window]
[in a new window]
  		FIG. 1. Sequence analysis of a PRL phosphatase from T. cruzi. (A) The deduced amino acid sequence of T. cruzi PRL-1 (Tc, AY461711 [GenBank] , 176 amino acids) is compared to the PRL sequences of T. brucei (Tb, AAX69332 [GenBank] .1, 176 amino acids), Homo sapiens (Hs, NP_003454 [GenBank] .1, 173 amino acids), Caenorhabditis elegans (Ce, AAC17103 [GenBank] .1, 190 amino acids), and Drosophila melanogaster (Dm, AAF53506 [GenBank] .2, 176 amino acids). Identical and conserved residues are shown in black and gray boxes, respectively. The active site is in an open box. The asterisk denotes the Cys107 essential for phosphatase activity. The sequence used to raise antibodies (Pep TcPRL-1) is underlined, and the CAAX motif is also shown. (B) Percent identity between the sequences shown in panel A.

 
Genomic organization, mRNA expression, and Western blot analysis of PRL-1 in T. cruzi. Genomic DNA from CL epimastigotes was digested with several restriction enzymes and hybridized with a labeled coding region of TcPRL-1 as a probe. Digestion with HincII, PstI, and MluI, enzymes that do not cut inside the ORF, rendered several bands in Southern blots (Fig. S1 in the supplemental material). HaeIII cut three times inside the ORF but rendered fragments of ca. 150 to 180 bp that are not detected in a 0.8% agarose gel. Therefore, the bands detected in the gel after HaeIII digestion correspond to the C-terminal part of the gene from different copies (Fig. S1 in the supplemental material, lower panel). The same holds true for HinfI. These results suggest that TcPRL-1 belongs to a gene family composed by several members in T. cruzi. In fact, after a search of the TIGR databases (www.tigr.org) with TcPRL-1 as the query, we found four genomic sequences that contain paralogues to TcPRL-1. These sequences were aligned as shown in Fig. S2 in the supplemental material. The genomic sequence named chr_4784 is almost identical to TcPRL-1, while the rest of the sequences are between 90 and 97% identical to TcPRL-1 (Fig. S2B in the supplemental material).

Total RNA was extracted from epimastigotes (lane E), amastigotes (lane A), and tissue culture-derived trypomastigotes (lane T), and Northern blots of these RNAs were hybridized with the same probe used in Southern blot analysis. The same transcript of ~0.75 kb was observed in all of the life cycle stages of T. cruzi (Fig. 2A). However, if we compare the intensities of the bands between upper and lower panels (24S rRNA that was used to normalize the blot for equal loading), it might be inferred that in the amastigote form of the parasite the expression was slightly higher. Since regulation of gene expression in trypanosomatids is mainly posttranscriptional, the significance of this difference in RNA abundance is not as relevant as at the protein level. When cell lysates prepared from different stages were analyzed by Western blotting with polyclonal antiserum against recombinant TcPRL-1, a band of 21 kDa could be detected only in amastigotes (Fig. 2B). No signal was detected with preimmune serum (data not shown). However, these results do not rule out a low level of expression of TcPRL-1 in the other stages. In this regard, it has been reported before that in several cell lines PRL proteins are expressed at undetectable levels by Western blotting (11) or immunofluorescence (39) analysis.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 2. Expression analysis and purification of TcPRL-1. (A) Northern blot analysis of epimastigote (E), amastigote (A), and trypomastigote (T) stages of T. cruzi. Approximately 15 µg of total RNA were fractionated in a 1.5% formaldehyde-containing agarose gel, transferred to nylon membranes, and hybridized with the 32P-labeled probe used for Southern blot analysis (Fig. S1 in the supplemental material). To control for equal loading of RNA in each lane, the membranes were stripped and reprobed with a 32P-labeled T. cruzi 24S ribosomal probe (lower panel). (B) Total cell lysates from wild-type epimastigote (E), amastigote (A), and trypomastigote (T) stages of T. cruzi (30 µg of protein/lane) were separated by SDS-PAGE, transferred to nitrocellulose membranes, probed with anti-TcPRL-1 antibody (1:2,000) and horseradish peroxidase-conjugated secondary antibody (1:10,000), and visualized by chemiluminescence. Markers are in kilodaltons. (C) In the left panel, recombinant TcPRL-1 was obtained as a fusion with GST. GST-TcPRL-1 expression was only observed after IPTG induction (compare lane U [soluble extract from uninduced bacteria] versus lane I [soluble extract from induced bacteria]). After in-column digestion with thrombin (lane TD), a protein of 21 kDa corresponding to TcPRL-1 was obtained and, after glutathione addition, GST (27 kDa) was eluted (lane E). SDS-15% PAGE gel stained with Coomassie blue. Lanes U and I, 10 µg of protein. Lanes TD and E, 1 and 3 µg of protein, respectively. The right panel shows an SDS-15% PAGE gel loaded with one-third of the amount of protein used in the left panel, transferred to nitrocellulose, and incubated with anti-TcPRL-1 serum. Markers on the right are in kilodaltons.

 
TcPRL-1 is an active tyrosine phosphatase. To demonstrate the phosphatase activity of TcPRL-1, we used recombinant protein (see Materials and Methods) in assays with p-NPP as substrate. Since the GST could potentially affect the enzymatic activity of TcPRL-1, this tag was removed by thrombin cleavage (Fig. 2C, left panel). The protein obtained after this treatment showed phosphatase activity (see below). The molecular mass of the recombinant form, ~21 kDa, is in agreement with that of PRLs from other organisms (5, 11, 38). The right panel in Fig. 2C shows that recombinant TcPRL-1 was effectively recognized by anti-TcPRL-1 specific antiserum raised against the C-terminal peptide underlined in Fig. 1A. The same result was obtained when antiserum raised against recombinant full-length TcPRL-1 was used (data not shown).

TcPRL-1 activity was linear with time until at least 1 h of reaction (Fig. 3A). Increasing enzyme concentration in the assay resulted in a proportional increase in the phosphatase activity (Fig. 3B). To determine the specificity of the reaction, we tested the activity of a mutant form of the enzyme where the Cys of the active site was changed to Ser (TcPRL-1C107S). As expected, this form showed no activity (Fig. 3B). A maximum activity level was detected at pHs between 7.5 and 8 (Fig. 3C). Like recombinant rat PRL-1 (11), TcPRL-1 has low activity under the conditions assayed (Fig. 3D). Recombinant TcPRL-1 showed a Michaelis-Menten behavior for p-NPP hydrolysis with a Km of 0.8 ± 0.05 µM p-NPP at pH 7.5. To determine whether TcPRL-1, as other PRLs, is a tyrosine phosphatase, we assayed the effect of different known inhibitors on its intrinsic activity. TcPRL-1 phosphatase activity was markedly inhibited by sodium o-vanadate in a dose-dependent manner, showing a 50% inhibition at 75 µM o-vanadate (Fig. 3E). Since PRLs show low similarity to dual specificity phosphatases (17, 38), we assayed the activity in the presence of several inhibitors to rule out a possible Ser/Thr phosphatase activity. The Ser/Thr phosphatase inhibitor okadaic acid (up to 1 mM), tested under similar conditions, showed no inhibitory effect against TcPRL-1 (Fig. 3F). Similar results were obtained with NaF (up to 5 mM) and EDTA (up to 10 mM).



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 3. Characterization of the phosphatase activity of TcPRL-1 with p-NPP or phosphorylated MBP as substrate. (A) Time-dependent hydrolysis of p-NPP assayed as described in Materials and Methods. (B) Enzyme concentration dependency using TcPRL-1 (continuous line) or TcPRL-1C107S (dashed line). (C) Effect of pH on TcPRL-1 phosphatase activity. (D) Phosphatase activity in the presence of different concentrations of p-NPP (0-10 mM) was measured during 1 h with 140 µg of protein/ml. (E) Effect of sodium o-vanadate. The inhibition assay was performed by addition of different concentrations (0 to 1 mM) of sodium o-vanadate. (F) Effect of different inhibitors on TcPRL-1 activity. Abbreviations: o-Van, o-vanadate; OA, okadaic acid; PE, pentamidine. The data represent means ± the standard deviations of triplicate samples. (G) Increasing amounts of TcPRL-1 (0, 0.7, 1.4, and 2.1 µg of protein) or a fixed amount of TcPRL-1C107S (2.0 µg of protein) were incubated with [32P]MBP for 1 h at 37°C. Samples were then boiled and loaded onto a SDS-12% PAGE gel, run, dried, and exposed to autoradiography. Densitometric analysis of the protein bands is expressed as a percentage of the control value. The experiment was repeated three times with similar results.

 
A recent report (24) showed that PRLs are inhibited by pentamidine, an antiprotozoal drug, whose mechanism of action remains elusive. Pentamidine is effective against T. brucei (13) and amastigotes of L. mexicana (3). In inhibition assays, pentamidine showed little effect against recombinant TcPRL-1 (Fig. 3F) even at a concentration as high as 10 µg/ml.

In cells, PRL phosphatases act on phosphorylated proteins. We therefore assayed the activity of recombinant TcPRL-1 on MBP phosphorylated on tyrosine residues (see Materials and Methods). As shown in Fig. 3G, TcPRL-1 was able to dephosphorylate Tyr-P-MBP, in a concentration-dependent manner. No effect on MBP dephosphorylation was observed when the mutant form TcPRL-1C107S was used (Fig. 3G).

In vitro prenylation of TcPRL-1. The predicted amino acid sequence of TcPRL-1 contains the C-terminal CAAX motif with the sequence CAVM (Fig. 1A), suggesting that this protein might be posttranslationally modified with either a farnesyl or geranylgeranyl group. In T. cruzi, a PFT activity has been described (37). To examine whether TcPRL-1 is a substrate for a PFT activity, we carried out a prenylation assay with recombinant GST-TcPRL-1 fusion protein or a version lacking the last four amino acids (GST-TcPRL-1{Delta}CAVM) as prenyl acceptors and a T. cruzi epimastigote extract as a source of PFT activity. When labeled FPP was used as the isoprenoid donor, GST-TcPRL-1 was efficiently farnesylated (Fig. 4A). However, when geranylgeranyl pyrophosphate was used as donor, we were unable to detect any prenylation of GST-TcPRL-1, even after exposure of the gel for 2 weeks at –80°C (data not shown). Therefore, TcPRL-1 does not appear to be geranyl-geranylated in vitro. As expected, GST-TcPRL-1{Delta}CAVM was not prenylated with either isoprenoid donor, indicating that the last four amino acids are essential for this posttranslational modification (Fig. 4A and data not shown). Figure 4B shows a Coomassie blue-stained SDS-PAGE gel loaded with the samples used in the assay as a control for equal loading and the purity of the protein preparations.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 4. TcPRL-1 is farnesylated in vitro. Purified recombinant GST-TcPRL-1 and a mutant form without the prenylation motif (GST-TcPRL-1{Delta}CAVM) (2 µg each) were incubated with T. cruzi epimastigote extract and [3H]FPP as described in Materials and Methods. Radiolabeled proteins were analyzed by SDS-PAGE on a 15% gel, followed by fluorography. Prenylation of TcPRL-1 is shown in panel A. (B) Coomassie blue-stained SDS-PAGE of the recombinant proteins used in panel A. Markers on the right are in kilodaltons.

 
Expression of GFP-tagged TcPRL-1, TcPRL-1{Delta}CAVM, and TcPRL-1C107S{Delta}CAVM in T. cruzi. In general, the normal cellular levels of PRL proteins are low (11) and even undetectable by immunofluorescence analysis (39). Because of that and since more than one PRL exists (Fig. S1 and S2 in the supplemental material), we cloned an N-terminal epitope-tagged version of TcPRL-1 into the expression vector pRIBOTEX, and the resultant construct was used to transfect T. cruzi epimastigotes (see Materials and Methods). We fused GFP to the N terminus of TcPRL-1 in order to leave the C-terminal prenylation motif accessible for modification by PFT. To determine the presence and the expression level of the GFP-tagged proteins in the transfected parasites, we performed immunoprecipitations with anti-GFP serum using soluble extracts of the parasites transfected with the different constructs. Figure 5A shows that GFP, GFP-TcPRL-1, and GFP-TcPRL-1{Delta}CAVM were immunoprecipitated by anti-GFP serum. Fusion proteins were also detected when anti-TcPRL-1 serum was used in Western blot assays of extracts (Fig. 5B). In Fig. 5B, we used three times more extract of GFP-TcPRL-1-transfected parasites compared to those of the two other constructs to obtain a band of a similar intensity. A major protein band, with an apparent molecular mass of ~47 kDa, was detected in the transfected parasite population with anti-TcPRL-1 (Fig. 5B) and anti-GFP (Fig. 5A) sera. The lower expression levels observed in parasites expressing the wild-type (GFP-TcPRL-1) in comparison with the mutant constructs (GFP-TcPRL-1{Delta}CAVM or GFP-TcPRL-1C107S{Delta}CAVM) is in agreement with the immunofluorescence results shown below. The highest expression levels were observed with the double mutant (GFP-TcPRL-1C107S{Delta}CAVM). No growth defects were detected upon overexpression of these constructs.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 5. Immunodetection of proteins in epimastigotes stably transfected with wild-type or mutant forms of TcPRL-1. (A and B) T. cruzi epimastigotes were transfected with the pRIBOTEX constructs described in Materials and Methods and selected with 500 µg of Geneticin/ml in the growth medium. (A) Immunoprecipitation with anti-GFP-serum raised in rabbit and protein A-Sepharose. Samples were boiled and separated by SDS-PAGE. After transfer to a nitrocellulose membrane, the Western blot was developed with GFP antiserum raised in a mouse. E, extract used in the immunoprecipitation; IP, immunoprecipitation reaction. Anti-maltose-binding protein ({alpha}-MBP) serum was used as a control for specificity of the immunoprecipitation reaction with GFP-TcPRL-1 extract. (B) The same extracts used in panel A (10 µg) were subjected to SDS-PAGE, transferred to nitrocellulose, and incubated with anti-TcPRL-1 serum, raised against recombinant TcPRL-1. Three times more extract of TcPRL-1 (30 µg) was loaded to obtain a signal. Markers on the right are in kilodaltons.

 
TcPRL-1 is prenylated in vivo. To know whether prenylation of TcPRL-1 occurs in vivo and to determine its importance for the correct subcellular localization of the protein, we performed immunofluorescence microscopy studies with a GFP-tagged enzyme and its derivative mutants. Epimastigotes expressing a GFP-TcPRL-1 fusion protein exhibited punctuate staining near the posterior end of the cells in 93% ± 1% of the cells (n = 2, 50 cells analyzed) (Fig. 6B). In many cases, fluorescence was also observed in the anterior end near the flagellar pocket (Fig. 6B and 7B). Association of prenylated proteins with cellular membranes is usually dependent on their prenylation status. To test whether farnesylation was required for the localization of GFP-TcPRL-1 to the punctuate structures, we investigated its localization in the stably expressed mutant forms lacking the prenylation signal. Parasites expressing GFP-TcPRL-1{Delta}CAVM or GFP-TcPRL-1C107S{Delta}CAVM were mainly labeled in the cytoplasm (Fig. 6F and H), thus establishing that prenylation of GFP-TcPRL-1 is necessary for its association with intracellular membranes. Similar results were obtained when anti-GFP or anti-TcPRL-1 serum was used in indirect immunofluorescence studies (data not shown). Epimastigotes expressing GFP alone (Fig. 6D) exhibited strictly cytosolic labeling. Similar results were obtained with amastigotes. Amastigotes expressing GFP-TcPRL-1 fusion protein showed staining of a punctuate structure in their anterior region in 75% ± 5% of the cells (n = 2, 50 cells analyzed) (Fig. 6J) while those expressing GFP-TcPRL-1{Delta}CAVM showed cytosolic labeling (Fig. 6L). Analysis of individual cells in the GFP-TcPRL-1{Delta}CAVM populations of epimastigotes and amastigotes by fluorescence microscopy revealed different levels of expression, but the protein was always cytosolic. This argues against different localization because overexpression. Taken together, these results support the prenylation of TcPRL-1 in vivo, which is consistent with the in vitro data (Fig. 4), and that prenylation plays an important role in the subcellular localization of TcPRL-1. Interestingly, GFP-TcPRL-1, in contrast to mammalian PRL-1 (39), does not localize to the nucleus when unprenylated.



View larger version (58K):
[in this window]
[in a new window]
  		FIG. 6. Localization of GFP, GFP-TcPRL-1, and mutant forms by confocal fluorescence microscopy. Fluorescence images of T. cruzi epimastigotes (A to H) and amastigotes (I to L) are shown. B and J, GFP-TcPRL-1; D, GFP alone; F and L, GFP-TcPRL-1{Delta}CAVM; H, GFP-TcPRL-1C107S{Delta}CAVM. Panels A, C, E, G, I, and K are phase-contrast images of the same cells. Bar, 10 µm.

 


View larger version (44K):
[in this window]
[in a new window]
  		FIG. 7. Immunofluorescence microscopy showing the localization of TcPRL-1 in epimastigote (A and B), amastigote (C and D), and trypomastigote (E) forms of T. cruzi. The figure shows the colocalization of TcPRL-1 with cruzipain in epimastigotes (A) and partial colocalization of TcPRL-1 in epimastigotes (B), amastigotes (C and D), and trypomastigotes (E) with concanavalin A. (A) Fixed epimastigotes expressing GFP-TcPRL-1 were permeabilized and labeled with monoclonal antibody against cruzipain. Panels show (from left to right) phase-contrast microscopy, GFP view, cruzipain view, and overlay. (B and C) Epimastigotes (B) or amastigotes (C) expressing GFP-TcPRL-1 were incubated for 30 min with 5 µg of concanavalin A-TRITC/ml and then fixed. Panels show (from left to right) phase-contrast microscopy, GFP view, concanavalin A-TRITC view, and overlay. (D) Wild-type amastigotes were incubated with concanavalin A-TRITC and then fixed and reacted with anti-TcPRL-1 polyclonal antibody. Panels show (from left to right) phase-contrast microscopy, TcPRL-1 antibody view, concanavalin A-TRITC view, and overlay. DAPI staining was included in the overlay to show relationship of the staining pattern to the nucleus (arrow) and kinetoplast (arrowhead). (E) Y strain trypomastigotes expressing TcPRL-1 were incubated for 30 min with 5 µg of concanavalin A-TRITC/ml, fixed, and reacted with anti-TcPRL-1 polyclonal antibody. Panels show (from left to right) phase-contrast microscopy, TcPRL-1 antibody view, concanavalin A-TRITC view, and overlay. Bar, 10 µm.

 
Localization of TcPRL-1 in the endocytic pathway. The localization of TcPRL-1 close to the flagellar pocket and in punctuate structures in the posterior end of epimastigotes (Fig. 6B and 7B) suggested a localization in the endocytic pathway. Epimastigotes possess large membrane-bound organelles found in their posterior end that are known as reservosomes. Reservosomes are acidic organelles containing cruzipain (a cysteine proteinase) and ingested proteins, and it has been proposed that they are prelysosomal compartments (7, 10).

When epimastigotes expressing GFP-TcPRL-1 (Fig. 7A) were labeled with anti-cruzipain, they showed colocalization of the proteins in the posterior end of the cells, a finding consistent with the presence of GFP-TcPRL-1 in the reservosomes. Some GFP-TcPRL-1 labeling in the perinuclear region of the epimastigotes (GFP-TcPRL-1) might be indicative of localization in the endoplasmic reticulum due to protein overexpression. Concanavalin A has previously been used to label the flagellar pocket, cytostome, and endocytic pathway of trypanosomatids (9). When GFP-TcPRL-1-expresing epimastigotes (Fig. 7B) were incubated with a concanavalin A-TRITC (tetramethyl rhodamine isothiocyanate) conjugate, strong labeling was detected in the flagellar pocket region and other membranous structures near the anterior end of the cell as well as in the posterior reservosomes. The anterior GFP-TcPRL-1 partially colocalized with the concanavalin A-TRITC labeling, and the posterior GFP-TcPRL-1 colocalized with concanavalin A-TRITC labeling of the reservosomes.

To investigate the localization of TcPRL-1 in infective stages, late stationary phase wild-type, GFP-, GFP-TcPRL-1-, and GFP-TcPRL-1{Delta}CAVM-expressing epimastigotes were used to infect tissue culture cells (4), and the tissue cultures were analyzed for the presence of trypomastigotes and amastigotes. When GFP-TcPRL-1 expressing amastigotes were analyzed by fluorescence microscopy, a small region of fluorescence was noted (Fig. 7C) that partially colocalized with concanavalin A-TRITC. A similar pattern of TcPRL-1 distribution was noted in wild-type amastigotes reacted with anti-TcPRL-1 antibody (Fig. 7D), confirming that the GFP pattern was not due to mistargeting of the overexpressed protein. We also show in the right part of panel D the DAPI (4',6'-diamidino-2-phenylindole) staining of the nucleus (arrow) and kinetoplast (arrowhead) to rule out a kinetoplast localization of TcPRL-1. No staining was detected with preimmune serum (data not shown). Endocytosis of other markers such as transferrin or bovine serum albumin was not efficient in amastigotes, and the number of cells available prevented subcellular fractionation techniques to confirm the localization of TcPRL-1 in amastigotes.

The yield of trypomastigotes was low with the CL stock and, to rule out the possibility that overexpression of TcPRL-1 could be inhibiting transformation of amastigotes into trypomastigotes, we transfected Y strain epimastigotes with either vector alone or a plasmid expressing TcPRL-1. After differentiation of epimastigotes to infective forms (4, 21), we were able to obtain trypomastigotes in similar amounts to those obtained from cells transfected with vector alone (data not shown). TcPRL-1 expressing trypomastigotes also showed colocalization of the label with concanavalin A (Fig. 7E). No staining was detected with preimmune serum (data not shown).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report here that TcPRL-1, a gene encoding a functional PTP, is present in the T. cruzi genome. Comparison of the sequence of TcPRL-1 with those of other PTPs indicates that it is closely related to the family of PTPs known as PRLs. Although the overall identity with other eukaryotic PRL-1s is ca. 34 to 50% (Fig. 1), TcPRL-1 has several residues characteristic of PRLs (such as Ala114, Cys52, and Tyr56), as well as conserved residues involved in the catalytic mechanism that are common to all tyrosine phosphatases (Cys107, Arg113, and Asp75) (20).

The low phosphatase activity exhibited by TcPRL-1, when the synthetic substrate p-NPP (Fig. 3) is used, is in accordance to the nuclear magnetic resonance and kinetic study of Kozlov et al. (17). These authors proposed that the presence of an Ala following the active site, instead of a Ser/Thr, is responsible, in part, for the low activity of PRLs when compared with other tyrosine phosphatases. Another important feature is the distance between the Asp75 and the catalytic cysteine. Both residues might get in contact only after binding the protein substrate, explaining the low activity with p-NPP. Other residues might be responsible for the higher sensitivity to o-vanadate inhibition (50% inhibitory concentration = 75 µM versus 60% inhibition at 1 mM for rat PRL-1) and the absence of pentamidine inhibition of TcPRL-1 compared to mammalian PRL-1s (24).

Like other PRLs, the protein is farnesylated in its C-terminal region, and this modification is essential for its membrane localization. Posttranslational modification of proteins with farnesyl groups appears to be essential for localization of modified proteins to membranes and, consequently, for their biological function (40). Farnesyl groups can be transferred to cysteine residues within carboxy-terminal motifs present in several classes of proteins in a reaction catalyzed by a cytoplasmic PFT (40). Inhibition of protein farnesylation by substrate inhibitors of farnesyl transferases or by inhibitors of the isoprenoid synthesis pathway has a profound effect on cell morphology (25), cell replication (26), and intracellular signal transduction (18). Recent studies have indicated that protein farnesylation occurs in T. cruzi, since the growth of the intracellular forms is sensitive to protein farnesyl transferase inhibitors (37). Interestingly, growth of amastigotes of T. cruzi, the life stage that has higher expression of TcPRL-1 (Fig. 2B), was inhibited by CAAX mimetics and FPP analogs, with 50% inhibitory concentrations ranging from 10 to 50 µM. In contrast, T. cruzi epimastigotes were resistant to 50 to 100 µM PFT inhibitors (37). In addition to TcPRL-1, the only other protein that has been shown to be farnesylated in T. cruzi is TcRho 1, which is a small GTPase possibly involved in signal transduction and metacyclogenesis (8, 23). The identification of farnesylated proteins in T. cruzi will help to identify the mechanism of toxicity of isoprenoid synthesis and protein farnesylation inhibitors.

In epimastigotes, TcPRL-1 colocalizes in the reservosomes with the cysteine proteinase cruzipain. It also colocalizes with concanavalin A in the flagellar pocket, cytostome, and endocytic pathway of different stages. Reservosomes are large membrane-bound organelles found in the posterior end of T. cruzi epimastigotes. They are acidic, contain the cysteine protease cruzipain, and accumulate macromolecules ingested by the parasite through endocytic processes, such as albumin, peroxidase, transferrin, and low-density lipoprotein (10, 29, 30, 32). Reservosomes are considered a prelysosomal compartment (10). It has been suggested that they also contain lipids (31) and, since their number decreases during metacyclogenesis, they were postulated to have a role in the storage of nutrients and/or proteins necessary for this differentiation step (10). In amastigotes, TcPRL-1 localizes in a small region of the cells, which partially colocalizes with concanavalin A-TRITC staining. Attempts to induce uptake by amastigotes of other endocytic markers such as transferrin or bovine serum albumin failed, and the amount of amastigotes that could be obtained precluded the use of subcellular fractionation techniques to investigate this localization. Further work will be needed to investigate the localization of the endogenous protein in amastigotes.

The association of TcPRL-1 with membranes is abolished by the removal of the CAAX motif. Under this condition, TcPRL-1 localizes in the cytoplasm. In sharp contrast, mammalian PRLs, when unprenylated, shifted to the nucleus (39). A possible explanation for these observations is that mammalian PRLs have a nuclear localization signal near the C terminus. The C terminus of TcPRL-1 is the region of least similarity to other PRL-1s and does not show a signal for nuclear localization. The involvement of mammalian PRL-1 in cell proliferation is dependent on its nuclear localization. Therefore, the differential localization of unfarnesylated TcPRL-1 to the cytoplasm suggests that in unicellular eukaryotes this phosphatase must have a different role.

In conclusion, TcPRL-1 is the first PRL-type PTP characterized at the molecular level in unicellular eukaryotes and is localized to endocytic membranes of T. cruzi thanks to its C-terminal farnesyl modification.


   ACKNOWLEDGMENTS
 
We thank Iván D'Orso for careful reading the manuscript; Shuhong Luo for transfection and cloning of Y strain epimastigotes; Julio Scharfstein for the monoclonal antibody against cruzipain; and Berta Franke de Cazzulo, Liliana Sferco, and Linda Brown for technical assistance. Preliminary genomic data obtained from http://www.tigr.org was provided by the TIGR-SBRI-KI Sequencing Consortium supported by NIH grants AI45038, AI45061. and AI45039.

This study was supported in part by grants from the World Bank/United Nations Development Project/World Health Organization Special Program for Research and Training in Tropical Disease (TDR Project 970629), the Agencia Nacional de Promoción Cientifica y Tecnológica, the Carrillo-Oñativia Fellowship, the Ministerio de Salud, and the Fundación Antorchas, Argentina (to D.O.S.) and U.S. National Institutes of Health grant AI-23259 (to R.D.). This investigation was conducted in part in a facility constructed with support from Research Facilities Improvement Program Grant C06 RR 16515-01 from the National Center for Research Resources, U.S. National Institutes of Health. I.C.C. is a fellow of the Comisión de Investigaciones Científicas. D.O.S. is a researcher from the Consejo Nacional de Investigaciones Científicas y Técnicas.


   FOOTNOTES
 
* Corresponding author. Mailing address for Daniel O. Sánchez: Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín, Avenida General Paz y Albarellos, San Martín, Provincia de Buenos Aires 1650, Argentina. Phone: (5411) 4580-7285. Fax: (5411) 4752-9639. E-mail: dsanchez{at}iib.unsam.edu.ar. Mailing address for Roberto Docampo: Department of Cellular Biology, The University of Georgia, 621 Biological Sciences Building, Athens, GA 30602. Phone: (706) 542-8104. Fax: (706) 542-4271. E-mail: rdocampo{at}cb.uga.edu. Back

{dagger} Supplemental material for this article may be found at http://ec.asm.org/. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bakalara, N., A., Seyfang, T. Baltz, and C. Davis. 1995. Trypanosoma brucei and Trypanosoma cruzi: life cycle-regulated protein tyrosine phosphatase activity. Exp. Parasitol. 81:302-312.[CrossRef][Medline]
  2. Bakalara, N., A. Seyfang, C. Davis, and T. Baltz. 1995. Characterization of a life-cycle-stage-regulated membrane protein tyrosine phosphatase in Trypanosoma brucei. Eur. J. Biochem. 234:871-877.[Medline]
  3. Berman, J. D., and D. J. Wyler. 1980. An in vitro model for investigation of chemotherapeutic agents in leishmaniasis. J. Infect. Dis. 142:83-86.[Medline]
  4. Caler, E. V., S. V. de Avalos, P. A. Haynes, N. W. Andrews, and B. A. Burleigh. 1998. Oligopeptidase B-dependent signaling mediates host cell invasion by Trypanosoma cruzi. EMBO J. 17:4975-4986.[CrossRef][Medline]
  5. Cates, C. A., R. L. Michael, K. R. Stayrook, K. A. Harvey, Y. D. Burke, S. K. Randall, P. L. Crowell, and D. N. Crowell. 1996. Prenylation of oncogenic human PTP(CAAX) protein tyrosine phosphatases. Cancer Lett. 110:49-55.[CrossRef][Medline]
  6. Cool, D. E., and J. J. Blum. 1993. Protein tyrosine phosphatase activity in Leishmania donovani. Mol. Cell Biochem. 127/128:143-149.
  7. Cunha-e-Silva, N. L., G. C. Atella, I. A. Porto-Carreiro, J. A., Morgado-Diaz, M. G. Pereira, and W. de Souza. 2002. Isolation and characterization of a reservosome fraction from Trypanosoma cruzi. FEMS Microbiol. Lett. 214:7-12.[CrossRef][Medline]
  8. de Melo, L. D., J. L. Nepomuceno-Silva, C. Sant'Anna, N. Eisele, R. B. Ferraro, J. R. Meyer-Fernandes, W. de Souza, N. L. Cunha-e-Silva, and U. G. Lopes. 2004. TcRho1 of Trypanosoma cruzi: role in metacyclogenesis and cellular localization. Biochem. Biophys. Res. Commun. 323:1009-1016.[Medline]
  9. Denny, P. W., S. Gokool, D. G. Russell, M. C. Field, and D. F. Smith. 2000. Acylation-dependent protein export in Leishmania. J. Biol. Chem. 275:11017-11025.[Abstract/Free Full Text]
  10. de Souza, W. 2002. Basic cell biology of Trypanosoma cruzi. Curr. Pharmaceut. Design 8:269-285.[CrossRef][Medline]
  11. Diamond, R. H., D. E. Cressman, T. M. Laz, C. S. Abrams, and R. Taub. 1994. PRL-1, a unique nuclear protein tyrosine phosphatase, affects cell growth. Mol. Cell. Biol. 14:3752-3762.[Abstract/Free Full Text]
  12. Diamond, R. H., C. Peters, S. P. Jung, L. E. Greenbaum, B. A. Haber, D. G. Silberg, P. G. Traber, and R. Taub. 1996. Expression of PRL-1 nuclear PTPase is associated with proliferation in liver but with differentiation in intestine. Am. J. Physiol. 271:G121-G129.
  13. Docampo, R., and S. N. J. Moreno. 2003. Current chemotherapy of human African trypanosomiasis. Parasitol. Res. 90:S10-S13.
  14. Franke de Cazzulo, B. M., J. Martinez, M. J. North, G. H. Coombs, and J. J. Cazzulo. 1994. Effects of proteinase inhibitors on the growth and differentiation of Trypanosoma cruzi. FEMS Microbiol. Lett. 124:81-86.[CrossRef][Medline]
  15. Furuya, T., L. Zhong, J. R. Meyer-Fernandes, H.-G. Lu, S. N. J. Moreno, and R. Docampo. 1998. Ecto-protein tyrosine phosphatase activity in Trypanosoma cruzi infective stages. Mol. Biochem. Parasitol. 92:339-348.[CrossRef][Medline]
  16. Karin, M., and T. Hunter. 1995. Transcriptional control by protein phosphorylation: signal transmission from the cell surface to the nucleus. Curr. Biol. 5:747-757.[CrossRef][Medline]
  17. Kozlov, G., J. Cheng, E. Ziomek, D. Banville, K. Gehring, and I. Ekiel. 2004. Structural insights into molecular function of the metastasis-associated phosphatase PRL-3. J. Biol. Chem. 279:11882-11889.[Abstract/Free Full Text]
  18. Lerner, E. C., Y. Qian, M. A. Blaskovich, R. D. Fossum, A. Vogt, J. Sun, A. D. Cox, C. J. Der, A. D. Hamilton, and S. M. Sebti. 1995. Ras CAAX peptidomimetic FTI-277 selectively blocks oncogenic Ras signaling by inducing cytoplasmic accumulation of inactive Ras-Raf complexes. J. Biol. Chem. 270:26802-26806.[Abstract/Free Full Text]
  19. Martinez-Calvillo, S., I. Lopez, and R. Hernandez. 1997. pRIBOTEX expression vector: a pTEX derivative for a rapid selection of Trypanosoma cruzi transfectants. Gene 199:71-76.[CrossRef][Medline]
  20. Mertz, L. M., and A. Rashtchian. 1994. Nucleotide imbalance and polymerase chain reaction: effects on DNA amplification and synthesis of high specific activity radiolabeled DNA probes. Anal. Biochem. 221:160-165.[CrossRef][Medline]
  21. Montalvetti, A., P. Rohloff, and R. Docampo. 2004. A functional aquaporin colocalizes with the vacuolar proton pyrophosphatase to acidocalcisomes and the contractile vacuole complex of Trypanosoma cruzi. J. Biol. Chem. 279:38673-38682.[Abstract/Free Full Text]
  22. Murta, A. C., P. M. Persechini, T. Souto-Padron, W. de Souza, J. A. Guimaraes, and J. Scharfstein. 1990. Structural and functional identification of GP57/51 antigen of Trypanosoma cruzi as a cysteine proteinase. Mol. Biochem. Parasitol. 43:27-38.[CrossRef][Medline]
  23. Nepomuceno-Silva, J. L., K. Yokoyama, L. D. de Mello, S. M. Mendonca, J. C. Paixao, R. Baron, J. C. Faye, F. S. Buckner, W. C. Van Voorhis, M. H. Gelb, and U. G. Lopes. 2001. TcRho1, a farnesylated Rho family homologue from Trypanosoma cruzi: cloning, trans-splicing, and prenylation studies. J. Biol. Chem. 276:29711-29718.[Abstract/Free Full Text]
  24. Pathak, M. K., D. Dhawan, D. J. Lindner, E. C. Borden, C. Farver, and T. Yi. 2002. Pentamidine is an inhibitor of PRL phosphatases with anticancer activity. Mol. Cancer Ther. 1:1255-1264.[Abstract/Free Full Text]
  25. Prendergast, G. C., J. P. Davide, S. J. DeSolms, E. A. Giuliani, S. L. Graham, J. B. Gibbs, A. Oliff, and N. E. Kohl. 1994. Farnesyltransferase inhibition causes morphological reversion of ras-transformed cells by a complex mechanism that involves regulation of the actin cytoskeleton. Mol. Cell. Biol. 14:4193-4202.[Abstract/Free Full Text]
  26. Quesney-Huneeus, V., M. H. Wiley, and M. D. Siperstein. 1979. Essential role for mevalonate synthesis in DNA replication. Proc. Natl. Acad. Sci. USA 76:5056-5060.[Abstract/Free Full Text]
  27. Ramirez, M. I., L. M. Yamauchi, L. H. de Freitas, Jr., H. Uemura, and S. Schenkman. 2000. The use of the green fluorescent protein to monitor and improve transfection in Trypanosoma cruzi. Mol. Biochem. Parasitol. 111:235-240.[Medline]
  28. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  29. Scott, D. A., R. Docampo, J. A. Dvorak, S. Shi, and R. D. Leapman. 1997. In situ compositional analysis of acidocalcisomes in Trypanosoma cruzi. J. Biol. Chem. 272:28020-28029.[Abstract/Free Full Text]
  30. Soares, M. J., and W. de Souza. 1991. Endocytosis of gold-labeled proteins and LDL by Trypanosoma cruzi. Parasitol. Res. 77:461-468.[CrossRef][Medline]
  31. Soares, M. J., M. F. de Souza, and W. de Souza. 1987. Ultrastructural visualization of lipids in trypanosomatids. J. Protozool. 34:199-203.[Medline]
  32. Soares, M. J., T. Souto-Padrón, and W. de Souza. 1992. Identification of a large pre-lysosomal compartment in the pathogenic protozoon Trypanosoma cruzi. J. Cell Sci. 102:157-167.[Abstract/Free Full Text]
  33. Tonks, N. K., and B. G. Neel. 2001. Combinatorial control of the specificity of protein tyrosine phosphatases. Curr. Opin. Cell Biol. 13:182-195.[CrossRef][Medline]
  34. Wang, J., C. E. Kirby, and R. Herbst. 2002. The tyrosine phosphatase PRL-1 localizes to the endoplasmic reticulum and the mitotic spindle and is required for normal mitosis. J. Biol. Chem. 277:46659-46668.[Abstract/Free Full Text]
  35. Werner, S. R., P. A. Lee, M. W. DeCamp, D. N. Crowell, S. K. Randall, and P. L. Crowell. 2003. Enhanced cell cycle progression and down regulation of p21Cip1/Waf1 by PRL tyrosine phosphatases. Cancer Lett. 202:201-211.[CrossRef][Medline]
  36. Yarovinsky, T. O., D. W. Rickman, R. H. Diamond, R. Taub, G. S. Hageman, and C. Bowes Rickman. 2000. Expression of the protein tyrosine phosphatase, phosphatase of regenerating liver 1, in the outer segments of primate cone photoreceptors. Brain Res. Mol. Brain Res. 77:95-103.[Medline]
  37. Yokoyama, K., P. Trobridge, F. S. Buckner, J. Scholten, K. D. Stuart, W. C. Van Voorhis, and M. H. Gelb. 1998. The effects of protein farnesyltransferase inhibitors on trypanosomatids: inhibition of protein farnesylation and cell growth. Mol. Biochem. Parasitol. 94:87-97.[CrossRef][Medline]
  38. Zeng, Q., W. Hong, and Y. H. Tan. 1998. Mouse PRL-2 and PRL-3, two potentially prenylated protein tyrosine phosphatases homologous to PRL-1. Biochem. Biophys. Res. Commun. 244:421-427.[CrossRef][Medline]
  39. Zeng, Q., X. Si, H. Horstmann, Y. Xu, W. Hong, and C. J. Pallen. 2000. Prenylation-dependent association of protein-tyrosine phosphatases PRL-1, -2, and -3 with the plasma membrane and the early endosome. J. Biol. Chem. 275:21444-21452.[Abstract/Free Full Text]
  40. Zhang, F. L., and P. J. Casey. 1996. Protein Prenylation: molecular mechanisms and functional consequences. Annu. Rev. Biochem. 65:241-269.[CrossRef][Medline]
  41. Zingales, B., M. E. Pereira, R. P. Oliveira, K. A. Almeida, E. S. Umezawa, R. P. Souto, N. Vargas, M. I. Cano, J. F. da Silveira, N. S. Nehme, C. M. Morel, Z. Brener, and A. Macedo. 1997. Trypanosoma cruzi genome project: biological characteristics and molecular typing of clone CL Brener. Acta Trop. 68:159-173.[CrossRef][Medline]

Eukaryotic Cell, September 2005, p. 1550-1561, Vol. 4, No. 9
1535-9778/05/$08.00+0     doi:10.1128/EC.4.9.1550-1561.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


<
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cuevas, I. C.
Right arrow Articles by Docampo, R.
Right arrow Search for Related Content