Identification and Characterization of an Unusual Double Serine/Threonine Protein Phosphatase 2C in the Malaria ParasitePlasmodium falciparum *

We have cloned a gene from Plasmodium falciparum with homology to the Mg2+-dependent serine/threonine protein phosphatase 2C (PP2C) family. The predicted coding region is 920 amino acids long, twice the size of other members of this family. We show that this protein can be divided into two halves (Pf2C-1 and Pf2C-2), each a complete phosphatase unit with homology to other phosphatases of this class. To study the function of this PP2C, we have tested the ability of different constructs to complement conditional null mutants of yeast. Our results show that expression of the full-length protein, the first half alone, the second half alone, or a hybrid with the N terminus of the first half and the C terminus of the second half was able to complement the heat shock response defect of aSchizosaccharomyces pombe strain with a PP2C (PTC1) deletion. Recombinant P. falciparum PP2C expressed in Escherichia coli was active in dephosphorylating 32P-labeled casein in an Mg2+- or Mn2+-dependent reaction. Each half alone was also active in recombinant form. Using the two-hybrid system, we have shown that the two halves can interact. Gel filtration assay of P. falciparum protein extracts suggests that full-length PfPP2C is a dimer, and phosphatase activity competition experiments indicate that dimerization of PfPP2C is required for its optimal activity. This unusual phosphatase molecule appears to be composed of four catalytic units on two polypeptide chains.

Two major families of protein phosphatases (phosphoprotein phosphatase or PPP and Mg 2ϩ -dependent protein phosphatase or PPM) catalyze the dephosphorylation of serine/threonine residues (1)(2)(3). In both eucaryotes and procaryotes, the Mg 2ϩdependent protein phosphatases are a large and varied family whose defining member is PP2C. 1 PP2C is present in cells at lower levels than other members of the serine/threonine phosphatase family (4). From bacteria to mammals, PP2C is a monomeric protein with a molecular mass range of 40 -50 kDa and requires divalent metal ions (Mg 2ϩ or Mn 2ϩ ) for its activity (5).
It is becoming clear that the PP2C proteins are involved in regulating stress response pathways in both procaryotes and eucaryotes. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, the PP2C genes PTC1 and PTC3 were shown to be involved in the regulation of protein kinase cascades that are induced in response to heat shock or osmotic changes (6,7). In mammalian hepatocytes, PP2C reverses, by dephosphorylating the AMP-activated protein kinase, the inhibition of cholesterol and fatty acid biosynthesis (8). Similarly, in Bacillus subtilis, the protein phosphatase SpoIIE regulates the steadystate level of phosphorylation of SpoIIAA, an anti-transcription factor, to control sporulation in response to ADP/ATP ratios (9,10). WIP1, a human PP2C-like gene, has been isolated as a transcript that is highly expressed in response to ionizing radiation (11).
Some proteins in the Mg 2ϩ -dependent protein phosphatase family contain, in addition to a catalytic region, a domain involved in other physiological functions. Thus, the N-terminal region of ABI1 in Arabidopsis thaliana contains a consensus sequence for a calcium-binding EF-hand (12,13). This domain may gauge the level of calcium in the cytoplasm in response to abscisic acid hormone and determine the level of phosphorylated proteins required for the maintenance of the cell cycle or stomatal aperture (13). SpoIIE of B. subtilis contains a region with 10 membrane-spanning segments (9,10), and the S. cerevisiae adenylate cyclase contains an upstream region of 300 amino acids that shares sequence similarity with PP2C (14).
This work describes the isolation of a PP2C-like protein, PfPP2C, from the malaria parasite Plasmodium falciparum. Each year, P. falciparum is estimated to kill between 1.5 and 2.7 million people, the majority of whom are children under five (15). Because the parasite and mosquitoes have developed resistance to drugs and pesticides, the identification of new targets for prophylaxis and treatment is urgently needed. The P. falciparum PP2C is composed of two active phosphatases fused together. The two units of PfPP2C complement a yeast deletion mutant, suggesting that this protein, like its homologues, may be active in response to osmolarity changes or other stress. PfPP2C may play a role in protecting the parasite against changes in the erythrocyte environment during infection. tions, and hybridization preparations were performed as described by Maniatis et al. (17).
Transformation-Yeast transformation was performed as described by Elble (18).
Protein Immunoblotting-Infected red blood cells from a 12-ml plate were treated with 0.1% saponin in phosphate-buffered saline for 30 min at 37°C. After centrifugation at 1875 ϫ g for 10 min, the pellet was washed in phosphate-buffered saline and resuspended in 500 l of phosphate-buffered saline. After sonication, the samples were heated for 10 min at 95°C and loaded onto 7.5% SDS-polyacrylamide gels. After migration, proteins were transferred to nitrocellulose filters. Preincubation, antibody incubations, and washes were conducted in 10 mM Tris-Cl (pH 8), 150 mM sodium chloride, and 0.05% Tween 20 with 5% skim milk. A chemiluminescence kit (ECL, Amersham Pharmacia Biotech) was used to detect the immunological reaction.
Anti-PP2C Antibody Preparation-The two oligonucleotides O1 (CAG CGG ATC CCC AAT AGT GAA CAT ATG AAT ACT) and O2 (CGC TGG ATC CAC TAA TCA TTT TCT TTA TAA TTA TTA G) were used to amplify a sequence encoding 100 amino acids of PfPP2C. The fragment was then cloned into the BamHI site of pGEX-3X. After induction with 1 mM isopropyl-␤-D-thiogalactopyranoside, the GST-100 fusion protein was purified on glutathione-Sepharose beads, used to immunize rabbits in phosphate-buffered saline and Freund's complete adjuvant, and then boosted every 4 weeks using Freund's incomplete adjuvant. Blood was collected 10 days after the fourth boost. The serum was used at 1:10,000 dilution for immunoblots.
Protein Phosphatase Assay-Hydrolyzed and partially dephosphorylated bovine milk casein (Sigma) was labeled using [␥-32 P]ATP (6000 Ci/mmol) and the catalytic subunit of bovine heart protein kinase as recommended by the supplier. Radiolabeled casein was precipitated and washed twice with 20% trichloroacetic acid, washed once with acetone, dried, and suspended in 0.2 M Tris-HCl (pH 8.0) to 10 5 cpm/l. The phosphatase assay was performed as described by McGowan and Cohen (19) using 10 g of purified enzyme.
Gel Filtration Assay-Protein extracts were prepared from 12 ml of P. falciparum asynchronous culture (2% hematocrit, 10% parasitemia) and resuspended in 1 ml of sterile phosphate-buffered saline. After filtration, 200 l was run on Superose 12 HR 10/30 at 0.2 ml/min, and fractions of 500 l were collected. 20 l of each fraction was tested by Western blotting using anti-PfPP2C antibodies or by phosphatase activity assay in the presence of 10 mM Mg 2ϩ and 5 M okadaic acid.

RESULTS
Isolation of a New PP2C Homologue in P. falciparum-Using a cDNA library made from asexual stages of the red blood cell parasite P. falciparum, we isolated a full-length cDNA clone encoding a protein with homology to PP2C enzymes. The 3.7kilobase pair cDNA (GenBank™ accession number AF023665) has an open reading frame (PfPP2C) starting at position 391. The first ATG codon has a standard nucleotide sequence for translation initiation. PfPP2C is predicted to encode a protein of 920 amino acids with a molecular mass of 103 kDa, which is twice the size of other PP2C proteins. Homology searches conducted for the PfPP2C protein sequence revealed a significant similarity to PP2C proteins from different organisms. Computer alignments revealed that certain PP2C sequences in the data base matched best with the N-terminal part of PfPP2C (Pf2C-1), whereas others matched best the C terminus of PfPP2C (Pf2C-2). Fig. 1 shows the alignment of both Pf2C-1 and Pf2C-2 with PP2C proteins from different organisms. Conserved motifs needed for phosphatase function in other organisms (20) are conserved in the Plasmodium sequence, but otherwise, homology is remote. The two halves share distant homology, suggesting that PfPP2C may have resulted from the fusion of two ancestral phosphatases during evolution.
PfPP2C Expression during the Intraerythrocytic Cycle of the Parasite-To examine PfPP2C expression in P. falciparum, we generated polyclonal antibodies against an internal 100-amino acid portion of PfPP2C fused to GST. On a Western blot of parasite cell extract (Fig. 2), the immune serum (second through fourth lanes), but not the preimmune serum (first lane), recognized a ϳ103-kDa protein in all intraerythrocytic stages of the parasite.
PfPP2C Is a Double Serine/Threonine Protein Phosphatase-To test whether the full-length protein has a cation-dependent phosphatase activity, PfPP2C was fused to the Cterminal region of GST, and the fusion protein was purified and tested for phosphatase activity (Fig. 3A). Recombinant PfPP2C is active in the dephosphorylation of casein and is highly activated in the presence of Mg 2ϩ or Mn 2ϩ . The activity was minimally inhibited by 5 M okadaic acid, a compound effective in the blockage of type 1 and 2A phosphatases. Because PfPP2C is twice the size of normal PP2C proteins, and each half has homology to other PP2C proteins, we were interested in knowing if both halves can function independently. To test this, the first half alone (Pf2C-1), the second half alone (Pf2C-2), and a hybrid form with the N-terminal part of the first half and the C-terminal part of the second half (Pf2C-⌬) were expressed as GST fusion proteins in E. coli (Fig.  3B). The purified proteins were then assayed for phosphatase activity. The results in Fig. 3C show that each construct had phosphatase activity that was stimulated by Mg 2ϩ or Mn 2ϩ , suggesting that both halves and the hybrid form possess PP2C activities. The activity obtained with each half alone (Pf2C-1 or Pf2C-2) was substantially lower than that obtained with fulllength PfPP2C (Fig. 3A), suggesting that the presence of both phosphatases in the same molecule is necessary for optimal activity.
The Two Units (Pf2C-1 and Pf2C-2) Interact with Each Other-The fact that PfPP2C is composed of two fused phosphatases raised the question of whether these two units interact with each other. We used the two-hybrid system in S. cerevisiae, expressing Pf2C-1 downstream of the Gal4 DNA activation domain (pACTII-Pf2C-1) and Pf2C-2 or the hybrid form downstream of the Gal4-binding domain (pAS-CYH2-Pf2C-2 or pAS-CYH2-Pf2C-⌬, respectively) (21). The plasmids obtained were used to transform the yeast strain Y190, and the Leuϩ,Trpϩ transformants carrying both plasmids were tested for ␤-galactosidase expression using the X-gal filter lift assay (22,23). In transformants expressing each vector alone (data not shown) or coexpressing pACTII-Pf2C-1 and pAS-CYH2-Pf2C-⌬ (Fig. 4), no ␤-galactosidase expression was detected. In contrast, the transformants carrying pACTII-Pf2C-1 and pAS-CYH2-Pf2C-2 became blue in the presence of X-gal, indicating that the ␤-galactosidase gene was being transcribed and translated. This suggests that the two halves (Pf2C-1 and Pf2C-2) interact with each other.
The Dimerization of PfPP2C Is Necessary for Full Activity-Results from the two-hybrid system experiments suggest two hypotheses about the interaction between the two units (Pf2C-1 and Pf2C-2) in vivo. First, there could be an intramolecular association. Alternatively, PfPP2C may form dimers in which the Pf2C-1 of one polypeptide can interact with the Pf2C-2 of another. In an attempt to distinguish between these two possibilities, proteins from P. falciparum extracts were separated on a Superose 12 gel filtration column, and the fractions collected were then tested for phosphatase activity in the presence of Mg 2ϩ and okadaic acid. The results in Fig. 5 show a single peak of activity at an approximate molecular mass of 240 Ϯ 30 kDa, indicative of a dimer structure of PfPP2C. To confirm that the phosphatase activity of this peak corresponds to PfPP2C activity, all fractions (fractions 1-47) collected by gel filtration were tested by Western blotting using the anti-PfPP2C antibodies. The results in Fig. 5 show complete agreement between the Western blot and the phosphatase activity peaks, suggesting that the unique Mg 2ϩ -dependent phosphatase activity detected in this fraction corresponds to PfPP2C.
We have further explored subunit and domain interactions by mixing purified components and assessing activity (Fig. 6). PfPP2C has higher specific activity than either half alone. When the two halves are mixed together, there is no stimulation of activity (data not shown). When PfPP2C is mixed with Pf2C-1 or Pf2C-2, the activity decreases 5-6-fold from the level of PfPP2C alone, approaching the activity of the isolated halves.
PfPP2C Can Function in a Heterologous Stress Response-To test whether PfPP2C is a functional homologue of yeast PP2C, we tested the effect of PfPP2C expression in a PTC1-deleted strain of S. pombe. The PTC1 gene encodes a PP2C that is proposed to play an important role in the yeast osmolarity response. The deletion strain has a greatly reduced ability to survive exposure to elevated temperature; the cells die more quickly after shifting from 25 to 48°C than do wild-type cells (24). We made a construct in which PfPP2C is expressed under the control of the S. cerevisiae GAL1 or ADH promoter or under the control of the S. pombe nmt promoter. Both S. cerevisiae promoters are constitutive in S. pombe. The plasmids obtained were then used to transform the KS834 strain deleted for the PTC1 gene. The transformants were first tested by Western blotting and showed expression of PfPP2C (data not shown). They were then tested for growth at 37°C and for their ability to survive after shifting from 25 to 48°C with or without  3). B, map of the GST fusion constructs made from PfPP2C. C, phosphatase activity of Pf2C-1, Pf2C-2, and Pf2C-⌬. The first half, second half, or the hybrid protein was expressed and purified as a GST fusion protein in E. coli. 10 g of each purified protein was tested for phosphatase activity in the absence of cations (group 1), with 10 mM Mg 2ϩ (group 2), or with 10 mM Mn 2ϩ (group 3). pretreatment at 37°C. Similar results were obtained with all three promoters; data are shown only for the GAL1 promoter construct. The growth rate of the ⌬PTC1 strain expressing PfPP2C at 37°C was faster than that of the ⌬PTC1 strain carrying the vector alone, and its ability to form colonies after shifting directly from 25 to 48°C was similar to that of the wild-type strain (Fig. 7). This suggests that the malaria PP2C is able to play the same role as its homologue PTC1 in the stress response.
Pf2C-1, Pf2C-2, and Pf2C-⌬ constructs were also active in the yeast system. Each was able to partially correct the heat response defect of the ⌬PTC1 strain (Fig. 7). These results correlate with the in vitro activity data, indicating that PfPP2C is composed of two active phosphatases fused together and that their fusion is necessary for optimal activity within the cell. DISCUSSION We have isolated a new PP2C gene from P. falciparum called PfPP2C. In yeast, PP2C has been implicated in the response to extracellular stimuli such as osmolarity changes or heat shock. Such stresses are signaled through the MAPK cascade, consisting of Wik1, a MAPK kinase kinase that phosphorylates Wis1, a MAPK kinase, which in turn phosphorylates Spc1, a MAPK. Data in S. cerevisiae and S. pombe indicate that tyrosinespecific and serine/threonine phosphatases negatively regulate the stress-activated kinase cascades (6,(25)(26)(27). In both yeasts, genetic and biochemical studies are in agreement concerning the direct involvement of tyrosine-specific phosphatases in dephosphorylation of MAPK (Spc1 in S. pombe and Hog1p in S. cerevisiae). Genetic data also suggest that serine/threonine phosphatases negatively regulate the stress-activated MAPK cascades. 1) In S. cerevisiae, two PP2C genes (PTC1 and PTC3) were isolated as multicopy suppressors of mutations that caused hyperactivation of the Hog1p kinase cascade (6). 2) Mutations in the HOG1 gene suppress the synthetic lethality interaction involving ptp2 and ptc1 mutations (28). 3) In S. pombe, mutations in Spc1 (MAPK) or Wis1 (MAPK kinase) suppress the calcium hypersensitivity of mutants carrying deletions of two or three serine/threonine phosphatase genes (26). Biochemical data from Gaits et al. (7), however, indicate that in S. pombe, PTC1 does not negatively regulate Wis1 or Spc1. These authors suggest that PTC1 may act downstream of Spc1 kinase in the stress-activated signal transduction pathway by dephosphorylating an Atf1 cofactor involved in transcriptional induction (7).
In this study, we have shown by heterologous complementation that PfPP2C can play the same role as PTC1 in S. pombe and perhaps functions similarly in P. falciparum. We have also shown that PfPP2C is composed of two protein phosphatases (Pf2C-1 and Pf2C-2) fused together in the same polypeptide. Both Pf2C-1 and Pf2C-2 are active in vitro and are able to complement the heat shock stress response defect of a PTC1 deletion in S. pombe.
Most organisms have at least two PP2C genes. Often, different phosphatase 2C proteins are involved in the same signal transduction pathway. For example, in both S. cerevisiae and S. pombe, three genes encoding PP2C-like proteins are involved in the regulation of the MAPK cascade (6,26,28,29). Other genes have been isolated during the S. cerevisiae genome sequencing project encoding proteins that show homology to PP2C sequences, but as of yet have no function or activity assigned. In A. thaliana, both ABI1 and ABI2 are PP2C-like proteins involved in an abscisic acid-dependent stress response pathway (12,13,30).
In P. falciparum, we have evidence for only a single PP2C gene. Low stringency Southern and Northern blot analyses detect a single gene and transcript (data not shown), whereas gel filtration analysis reveals only a single activity peak eluting at a position expected of a molecule four times the size of a standard PP2C (Fig. 6). Our data are consistent with the notion that PfPP2C is a double phosphatase, wherein both halves possess a phosphatase fold. Purified PfPP2C, Pf2C-1, Pf2C-2, and a hybrid form composed of the first half of Pf2C-1 and the second half of Pf2C-2 are each active in dephosphorylating casein in vitro. A complete dimer appears to be necessary for maximal activity since the two halves alone or in combination possess considerably lower activity than the whole molecule, and either half interferes with the ability of the whole to achieve maximal activity upon mixing.
Taken together, these results suggest the model presented in Fig. 8, in which PfPP2C acts as dimer, and the Pf2C-1 of one polypeptide interacts with the Pf2C-2 of the second polypeptide. The whole dimer is, in effect, a tetrameric phosphatase.
Perhaps the unusual structure of PfPP2C can overcome the need in other organisms for additional genes, offering a novel way to regulate the signal transduction pathway. Study of the expression and activity of PfPP2C during stress conditions and analysis of its crystal structure could better our understanding of the mechanisms that monitor the stress response of the malaria parasite. Its unusual configuration is different enough from PP2C proteins of human cells to be considered a good target for new antimalarial drugs.