Raf kinase inhibitor protein affects activity of Plasmodium falciparum calcium-dependent protein kinase 1

Molecular & Biochemical Parasitology 151 (2007) 111–117

Raf kinase inhibitor protein affects activity of Plasmodium falciparum calcium-dependent protein kinase 1 Dominik Kugelstadt a , Dominic Winter b , Kirsten Pl¨uckhahn a , Wolf Dieter Lehmann b , Barbara Kappes a,∗ b

a Universit¨ atsklinikum Heidelberg, Abteilung f¨ur Parasitologie, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany Central Spectroscopy, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany

Received 8 December 2005; received in revised form 23 October 2006; accepted 24 October 2006 Available online 15 November 2006

Abstract Proteins, such as the raf kinase inhibitory protein (RKIP), serve as modulators of signalling pathways by either promoting or inhibiting the formation of productive signalling complexes through protein–protein interactions. In the present study, the plasmodial RKIP ortholog, PfPE-PB1, was cloned, recombinantly expressed and purified to homogeneity. The purified protein was used to investigate the effect of plasmodial RKIP on the autophosphorylation and substrate phosphorylation activity of Plasmodium falciparum calcium-dependent protein kinase 1, PfCDPK1. Phosphorylation of RKIP by PfCDPK1 in in vitro kinase assays suggests that RKIP may be an in vivo substrate of this kinase, although the specific activity of PfCDPK1 is approximately seven-fold lower when RKIP, instead of casein, an exogenous substrate of this enzyme, is used as a substrate. In addition to the observed phosphorylation of RKIP itself, its presence in the assays greatly enhanced the autophosphorylation capacity of PfCDPK1 by approximately 5.5-fold. This substantial increase in autophosphorylation activity was associated with a diminished substrate phosphorylation activity of PfCDPK1 when casein was used. At the same time, RKIP phosphorylation slightly increased when casein was included into the assays. Thus, RKIP is recognized as a substrate under in vitro conditions and appears to act as a regulator of PfCDPK1 kinase activity, which possibly is one of its actual functions in the parasite. © 2006 Elsevier B.V. All rights reserved. Keywords: Malaria; Plasmodium falciparum; Calcium-dependent protein kinase; PfCDPK1; Raf kinase inhibitor; RKIP

1. Introduction Ca2+ plays a central role in the biology of eukaryotic cells, serving a wide range of regulatory and signalling functions. There is substantial evidence of the importance of Ca2+ for the malaria parasite. External Ca2+ is required for both the invasion and development of the parasite within the red blood cell [1,2]. There is also a net uptake of Ca2+ by infected RBCs as the parasite matures [3]. Parasite development within the erythro-

Abbreviations: ␤-ME, ␤-mercaptoethanol; DTT, dithiothreitol; EDTA, ethylene diamine tetra acetic acid; IPTG, isopropyl 1-thio-␤-galactopyranoside; Ni-NTA, nickel-nitriloacetic acid agarose; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PfCDPK1, Plasmodium falciparum calcium-dependent protein kinase 1; PfRKIP, Plasmodium falciparum raf kinase inhibitor protein ∗ Corresponding author. Tel.: +49 6221 561774; fax: +49 6221 564643. E-mail address: [email protected] (B. Kappes). 0166-6851/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2006.10.012

cyte is inhibited by a range of Ca2+ ionophores, Ca2+ channel blockers and calmodulin antagonists [4,5]. The specific role of Ca2+ in the development of the intracellular parasite is not well understood, although Ca2+ -dependent, calmodulin-independent protein kinases appear to be key players in Ca2+ -mediated signal transduction processes and constitute the only Ca2+ -regulated protein kinase family present in the parasite [6,7]. The unique calmodulin-independent nature of these kinases is derived from a characteristic and highly conserved N-terminal serine/threonine protein kinase domain, which is contiguous with a C-terminal calmodulin-like Ca2+ -binding domain. CDPKs form multigene families in plants and certain protozoan species [8]. The genome of Plasmodium falciparum encodes a family of five classical CDPKs named PfCDPK1-5, sharing 39–56% amino acid identity and a CDPK related protein kinase [6,9–12]. Whereas the functions of PfCDPK1, 2 and 5 are unknown, the Plasmodium berghei equivalents of PfCDPK3 and PfCDPK4 have recently been identified as the molecular

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regulator of ookinete penetration of the layer covering the midgut epithelium [13] and the molecular switch that translates the xanthurenic acid-induced calcium signal into a cellular response by regulating cell cycle progression in the male gametocyte [6], respectively. In addition, the morphological differentiation from the immobile zygote to the mobile ookinete of Plasmodium gallinaceum has been attributed to a Ca2+ /calmodulin-dependent protein kinase; however, this is most likely a CDPK [14]. Raf kinase inhibitor protein, RKIP, was originally purified from bovine brain and was designated phosphatidylethanolamine-binding protein (PEBP), based on its ability to preferentially bind this particular class of phospholipids [15]. Subsequent studies revealed that RKIP/PEBP is a widely expressed and highly conserved protein with orthologs in bacteria, fungi, protozoa, plants and animals, which display a high degree of interspecies similarity [16]. Based on its capacity to bind phospholipids, it has been postulated that RKIP/PEBP functions as a lipid transporter in membrane biogenesis. However, analysis of the structure of RKIP/PEBP revealed that the binding pocket is too shallow to accommodate lipid tails [17], suggesting that the binding pocket could be used to bind phosphoproteins. Numerous functions of RKIP/PEBP proteins have now been reported. The yeast RKIP ortholog (Tfs1p) inhibits the vacuolar carboxypeptidase Y, and Ira2p, a yeast Ras GTPase-activating protein (GAP), attenuates Ras activation by causing hydrolysis of GTP [18,19]. In nematodes, RKIP is proposed to play a role in protection from host immune detection, and in Drosophila, expressing at least seven RKIP isoforms, these are thought to function in odorant binding [16]. In plants, RKIP/PEBP has been associated with switching from the vegetative to the reproductive growth phase, a key step in plant differentiation [20]. In mammals, RKIP/PEBP has been shown to inhibit serine proteases, specifically thrombin, neuropsin, and chymotrypsin, although it shares no homology with any of the known families of serine protease inhibitors [21]. The best known function of RKIP/PEBP in mammalian cells is as an inhibitor of the MAP kinase signalling pathway by suppression of Raf-1 kinase activity [22]. Binding of RKIP to Raf-1 abolishes the formation of the Raf1/MEK complex and thus prevents MEK phosphorylation and subsequent activation of the MEK/ERK pathway. Phosphorylation of RKIP on serine 153 by PKC triggers RKIP dissociation from Raf-1, allowing the formation of the Raf-1/MEK complex [23]. More recently, RKIP/PEBP was identified as a source of the neurostimmulatory peptide (HCNP) [24]. Aberrations in HCNP, or its precursor RKIP/PEBP, has been associated with certain neuropathologies, such as Alzheimer and Parkinson [25]. In the present study, we investigated the effect of plasmodial RKIP on the autophosphorylation and substrate phosphorylation activity of PfCDPK1. In vitro phosphorylation of RKIP by PfCDPK1 suggests that RKIP could be an in vivo substrate of the kinase. Whereas autophosphorylation of PfCDPK1 is strongly enhanced in the presence of PfRKIP, substrate phosphorylation is significantly reduced. Thus, our current data suggest that PfRKIP functions as a regulator of PfCDPK1 kinase activity in the parasite.

2. Materials and methods 2.1. Materials and parasite culturing P. falciparum HB3 and 3D7 were provided in house by professor Lanzer and cultivated according to Trager and Jensen in RPMI 1640 medium containing 5% human serum A+ under reduced oxygen [26]. 2.2. Cloning and RT-PCR of plasmodial RKIP The primary structure of plasmodial RKIP has been described as a putative phosphatidylethanolamine-binding protein by Trottein and Cowman (EMBL accession no: U18984) [27]. The full-length RKIP gene of P. falciparum was amplified by polymerase chain reaction (PCR) using the sequence-specific sense and antisense oligonucletotides PfRKIP.fwd (5 -TATTAAGGATCCATGACAATACCCACGATAAGTGAA-3 ) and PfRKIP.rev (5 -TTATTAGGATCCCTACCCTTCAATTTGGCACCATT-3 ) using P. falciparum HB3 genomic DNA as a template. The PCR fragments were cloned into the BamHI site of pBluescript II SK (Stratagene). For recombinant expression in pET-21a(+) (Novagen), an XhoI site was introduced at the 3 end of the gene, using the following primer: PfRKIP exp.rev (5 -TCAGAACTCGAGCCCTTCAATTTGGCCACCA-3 ). The nucleotide sequences of all constructs were confirmed by automated sequencing. For RT-PCR analysis, 3 × 108 blood stage parasites (strain 3D7) were used to isolate poly(A)+ RNA on oligo(dT) columns (Invitrogen). cDNA synthesis and amplification were performed in a two step PCR using random decamer primers (Ambion, Austin, TX) and subsequent standard PCRs with the genespecific primers RKIP XhoI.fwd (5 -AATAACTCG AGATGACAAT ACCCACGATA AGTGAAC-3 ) and RKIP BamHI.rev (5 -TAATAAGGAT CCCCCTTCAA TTTGGCACCA AT3 ) and PFD0720w.fwd (5 -TAATAACTCG AGATGGGAAA TAATTGCTGT GCAGGAAG-3 ) and PFD0720w.rev (5 TAATAAGAAT TCATCCGTTA GTCTCAATAA GAGAACATTG G-3 ). 2.3. Bacterial expression and purification of PfCDPK1 and PfRKIP PfCDPK1 was expressed in the E. coli strain SG13009 with induction by isopropyl 1-thio-␤-galactopyranoside (IPTG) [12] and purified as described in Zhao et al. [28]. For storage, glycerol was added to a final concentration of 10% and the protein was frozen at −80 ◦ C. Plasmodial RKIP was expressed in the E. coli strain BL21-CodonPlus® (DE3)-RIL (Stratagene). Cultures were grown in Luria broth supplemented with 100 ␮g ml−1 ampicillin and 34 ␮g ml−1 chloramphenicol until an OD600 of 0.8 was reached. Expression was induced by the addition of IPTG. Cells were allowed to grow for 3 h, were harvested by centrifugation, resuspended in lysis buffer (150 mM NaCl, 20 mM imidazole, 5 mM ␤-mercaptoethanol (␤-ME), 50 mM Tris–HCl, pH 7.6, 10% glycerol containing phenylmethyl-

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sulfonyl fluoride (120 ␮g ml−1 ), leupeptin (0.5 ␮g ml−1 ) and pepstatin A (0.7 ␮g ml−1 )), flash-frozen and stored at −80 ◦ C. The frozen pellet of 1 l of culture was thawed on ice; cells were disrupted by sonication, the suspension cleared by centrifugation (21,000 × g) and the crude supernatant applied to nickel-NTA (Qiagen). The column was successively washed with 600 mM NaCl, 5 mM ␤-ME, 50 mM Tris–HCl, pH 7.6 containing either 20 or 50 mM imidazole and eluted with the same buffer containing 200 mM imidazole. 2.4. Kinase assays Kinase assays were performed by 30 min incubations at 30 ◦ C in a final volume of 25 ␮l of a mixture containing 20 mM Tris–HCl, pH 7.4, 10 mM MgCl2 , 1 mM DTT, 87.5 ␮M ATP and 10 mM Ca2+ or 100 ␮g EGTA in the absence or presence of PfRKIP (3 ng–2.1 ␮g) and casein (0.5 mg ml−1 ) using 50 ng of the recombinant PfCDPK1. As recombinant plasmodial RKIP requires salt for its solubility, all kinase assays were performed in the presence of 28.3 mM NaCl. The PfCDPK1 stock used in these assays had a specific activity of 279 nmol−1 min−1 mg−1 protein determined with casein as the substrate. The reactions were initiated by the addition of either [␥-32 P]ATP or enzyme, and stopped by the addition of either SDS-PAGE sample loading buffer or 5 ␮l of 1.5 M H3 PO4 . The latter samples were processed as described in Ref. [28]. 2.5. Mass spectrometric identification of phosphorylation sites Protein bands were excised, cut into pieces, and treated as described earlier [29], but using digestion by chymotrypsin. Phosphopeptides were enriched from the mixture of proteolytic peptides using a commercial phosphopeptide isolation kit (Pierce, Rockford, IL) loaded with Ga(III). Mass spectra were recorded by using a hybrid Q-TOF mass spectrometer type Q-TOF 2 (Micromass, Manchester, UK). Spray capillaries were manufactured in-house by using a micropipette puller type P-87 (Sutter Instruments, Novato, CA) and coated with a semitransparent film of gold in a sputter unit type SCD 005 (BAL-TEC, Balzers, Liechtenstein). MS Data of the phosphopeptide enriched fractions were acquired in the automated MS to MS/MS switching mode. For each m/z value tandem MS spectra were recorded by using a mass-dependent set of five collision offsets to ensure optimal detection of both sequenceand composition-specific fragment ions.

Fig. 1. Presence of PfRKIP transcripts in parasite red blood cell stages. cDNA and genomic DNA from P. falciparum 3D7 was amplified at 35 PCR cycles. (1) PfRKIP PCR fragment from cDNA (573 bp), (2) PfRKIP PCR fragment from genomic DNA (573 bp), (3) PFD0720w PCR fragment from cDNA (890 bp), (4) PFD0720w PCR fragment from genomic DNA (2175 bp). Since, in contrast to the PFD0720w, the PfRKIP gene does not contain introns, reaction (3) served as a control to ensure that the cDNA preparation used was not contaminated with genomic DNA.

sorbitol synchronized parasites was isolated and analyzed by RTPCR (Fig. 1). RT-PCR amplification resulted in a fragment of the expected size of 573 bp (Fig. 1, lane 1). Since RKIP does not contain introns (Fig. 1, lane 2), a control PCR was performed to rule out genomic DNA contamination of the cDNA. Amplification of PFD0720, our control gene, resulted in a single fragment of 890 bp, the expected size of the PFD0720 cDNA (Fig. 1, lane 3). In contrast, amplification of PFD0720 on genomic DNA using the same primer combination generated a fragment of 2175 bp, corresponding to the expected gene size of PFD0720. 3.2. Recombinant expression and purification of PfRKIP For recombinant expression of RKIP, BamHI and XhoI sites were introduced at the 5 and 3 end of RKIP and the respective fragment cloned into the expression vector pET-21a(+). Expression using this vector produces a protein with a C-terminal (His)6 -tag. The major fraction of the recombinant protein was insoluble and accumulated in inclusion bodies (data not shown). However, purification on a Ni-NTA column resulted in approximately 40–70 ␮g of soluble RKIP per litre of bacterial culture. The affinity-purified plasmodial RKIP was dialysed against kinase assay buffer containing 150 mM NaCl.

3. Results 3.1. PfPEBP/RKIP is expressed in parasite blood stages

3.3. RKIP is a substrate of PfCDPK1 and enhances the autosphosphorylation activity of the kinase

The microarray studies performed by Le Roch et al. [30] and Bozdech et al. [31] suggested that the plasmodial RKIP gene is expressed at very low levels hardly detectable in blood stage parasites. To determine whether plasmodial RKIP is indeed transcribed in erythrocytic stages of P. falciparum, mRNA from

As the plasmodial RKIP precipitated in sodium free buffer, all kinase assays were performed at a final sodium chloride concentration of 28.3 mM. After the kinase reaction, samples were separated on a 12% SDS-PAGE and analyzed by autoradiography.

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Examination of the autoradiographs revealed that RKIP significantly activated the autophosphorylation capacity of PfCDPK1 (Fig. 2A, lanes 2 and 4). To quantify the effect of RKIP concentration on the stimulation of PfCDPK1 autophosphorylation activity, increasing quantities of RKIP (3–2100 ng) were added to the kinase assays. Reactions were separated on SDS-PAGE, the PfCDPK1 bands cut out of the gel and the incorporated radioactivity determined. RKIP led to a maximal increase of PfCDPK1 autophosphorylation activity of 5.5-fold (Fig. 2C). This plateau was reached with a RKIP concentration of 900 ng, equivalent to a ∼50-fold molar excess of RKIP (Fig. 2C). Half-maximal stimulation of PfCDPK1 autophosphorylation activity was achieved with a RKIP concentration of 100 ng, corresponding to a roughly 5.5-fold molar excess of the protein (Fig. 2C). 3.4. PfRKIP decreases the substrate phosphorylation activity of PfCDPK1 As altered autophosphorylation behaviour of a kinase often affects its substrate phosphorylation activity, we investigated whether addition of RKIP to the kinase assay had an effect on the phosphorylation of casein, an exogenous substrate of PfCDPK1 (Fig. 3A). To determine the radioactivity incorporated into the individual bands, we proceeded as described above. RKIP phosphorylation increased by a factor of 1.6 when casein was present in the kinase reaction (Fig. 3A, lane 3; data not shown). However, in the same reaction, phosphorylation of

Fig. 2. RKIP is a substrate of PfCDPK1 and affects its autophosphorylation activity. (A) Autoradiograph showing RKIP phosphorylation and autophosphorylation of PfCDPK1. PfCDPK1 in the absence (1) and presence (3) of Ca2+ , and plasmodial RKIP and PfCDPK1 in the absence (2) and presence (4) of Ca2+ . (B) Substrate phosphorylation activity of PfCDPK1 as a function of RKIP concentration (). The phosphorylation of RKIP at a concentration of 2.1 ␮g was set to 100%. (C) Autophosphorylation activity of PfCDPK1 as a function of RKIP concentration (䊉). Autophosphorylation in the absence of PfRKIP was set to 100%. Data presented in (B and C) represent the mean ± S.D. of six independent determinations.

PfCDPK1 recognized RKIP as a substrate (Fig. 2A, lanes 2 and 4). Although a residual phosphorylation of the protein was observed in the absence of Ca2+ (Fig. 2A, lane 2), addition of Ca2+ strongly increased the phosphorylation of RKIP by a factor of 5.4 (Fig. 2A, lane 4). In the presence of Ca2+ , RKIP phosphorylation steadily increased until a concentration of 1.6 ␮M (equivalent to 900 ng) was reached (Fig. 2B).

Fig. 3. RKIP alters the substrate phosphorylation activity of PfCDPK1. (A) Autogradiograph showing the phosphorylation of casein (lane 1), plasmodial RKIP (lane 2) and casein in the presence of RKIP (3). (B) Substrate phosphorylation activity of PfCDPK1 towards casein as a function of RKIP concentration (). The phosphorylation of casein in the absence of RKIP was set to 100%. Data represent the mean ± S.D. of six independent determinations.

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Fig. 4. NanoESI mass spectra of the peptides VVpSGIKTKEL isolated from a digest of phosphorylated PfRKIP by Ga(III)-IMAC; (a) survey spectrum of the phosphopeptide-enriched fraction; (b) tandem mass spectrum of the ion at m/z 577.3. The survey spectrum shows an abundant signal for the molecular ion of the phosphopeptide VVpSGIKTKEL, which is identified by the characteristic phosphate- and sequence-specific fragment ions of its tandem mass spectrum.

casein decreased by approximately 60% (Fig. 3A, compare lanes 1 and 3). To quantify the effect of RKIP concentration on the inhibition of the casein phosphorylation activity of PfCDPK1, increasing quantities of RKIP (3–2100 ng) were added to the kinase assays. Inhibition of casein phosphorylation was dependent on the RKIP concentration (Fig. 3B). Its phosphorylation continuously decreased, reaching half-maximal inhibition of casein phosphorylation of 40% at a 10-fold excess of casein and maximum inhibition of 80% at approximately four-fold excess of casein. 3.5. PfCDPK1 phosphorylates serine 96 of plasmodial RKIP Phosphorylation of RKIP by PKC on S153 results in a dissociation of RKIP from Raf-1, leading to an activation of the MAP kinase pathway [32]. As S153 is not conserved in the plasmodial RKIP ortholog, phosphorylated RKIP was subjected to Q-TOF nanoESI tandem mass spectrometric analysis. The result of the analysis is shown in Fig. 4. Phosphorylation occurred on a peptide spanning positions 95–103 in the plasmodial RKIP amino acid sequence, with the phosphorylated residue being S96 . Determination of this phosphorylation site revealed a thus far unknown phosphorylation recognition motif of PfCDPK1, namely S/TXXK/R. 4. Discussion The study presented here provides evidence that RKIP could be a modulator of PfCDPK1 kinase activity in the P. falci-

parum parasite. The presence of plasmodial RKIP increased the Ca2+ -independent, as well as Ca2+ -dependent autophosporylation activity of the kinase (Fig. 2A, lanes 2 and 4 and Fig. 2C). This is remarkable, since neither the autophosphorylation of Raf1 nor of protein kinase C is altered by RKIP in the mammalian system [22,32]. In mammals, RKIP is a substrate of PKC, not of Raf-1 [22,32]. However in the plasmodial system, RKIP is a substrate of PfCDPK1, which belongs to a different protein kinase family than PKC (Fig. 2A and B). Further evidence that RKIP could be a modulator of PfCDPK1 activity in the parasite is provided by the fact that the enhanced autophosphorylation activity induced by RKIP is associated with a diminished substrate phosphorylation activity when casein is used as an exogenous substrate (Fig. 3A). Under our assay conditions, where plasmodial RKIP concentrations ranged from 3 ng to 2.1 ␮g, the substrate phosphorylation activity of PfCDPK1 towards casein decreased continuously in a concentration-dependent manner, resulting in a maximum inhibition of 80% (Fig. 3B). The inhibition of casein phosphorylation cannot simply be explained by competition of the two substrates, RKIP and casein, for the substrate binding site of the kinase since this would lead to a decrease in phosphorylation of both proteins. Plasmodial RKIP phosphorylation, in this case, increased in the presence of casein by about 1.6-fold (Fig. 3A, lane 3; data not shown). Whether this slight increase is indeed significant and of biological relevance has to be established. In the mammalian system, the substrate phosphorylation activity of Raf-1 towards myelin basic protein was not altered by binding of RKIP (up to concentrations of 5 ␮M of RKIP in the kinase assay corresponding to the 1.8 ␮g concentration in our

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assays) [22]. Whether the substrate phosphorylation activity of PKC towards exogenous substrates is affected by the presence of RKIP is unknown. Protein kinase C phosphorylates RKIP at S153 [32]. As the amino acid S153 is not conserved in the plasmodial ortholog, we searched for other putative phosphorylation sites within the plasmodial RKIP sequence that could be recognized by PfCDPK1. Using Q-TOF nanoESI tandem mass spectrometric analysis, S96 was identified as the phosphorylated amino acid (Fig. 4). Phosphorylation of S96 follows the phosphorylation recognition motif S/TXXK/R, which has also been described for other CDPKs [8] and has now been determined as a phosphoacceptor peptide sequence of PfCDPK1 for the first time. PfCDPK1 is expressed throughout the whole intraerythrocytic development of the parasite with highest protein levels found in ring and schizont stages [28]. The kinase is predominantly found in extracellular compartments of the P. falciparum parasite, where dynamic changes of the membranes, such as growth of the parasitophorous vacuole membrane and construction and remodelling of the tubovesicular network, are taking place [33]. In contrast to PfCDPK1, RKIP was originally described as a cytosolic protein [15]. Binding studies revealed its affinity for phosphatidylethanolamine, a phospholipid mainly located on the inner leaflet of the plasma membrane, suggesting an additional localization of RKIP at this cellular location [34]. However, such a localization would exclude a possible interaction of PfCDPK1 and RKIP in the parasite, since during most of the intraerythrocytic development of Plasmodium (ring to schizont stage), PfCDPK1 is found in “extracellular compartments” [33]. Studies by Hengst et al. revealed that, despite having no obvious secretion signal, RKIP can be immunolocalized on the cell surface of rat fibroblasts and detected in conditioned medium [21]. Provided that the situation in P. falciparum is similar, PfCDPK1 and RKIP would be able to interact, since both are expressed in blood stages (Fig. 1, [28]). This is the first report on the effect of RKIP on a calciumdependent protein kinase. Whether RKIP is a modulator of PfCDPK1 in the parasite and what its precise role is will be addressed in further experiments. RKIP is a substrate for PfCDPK1, as it is for PKC, suggesting that PfCDPK1 may have a function similar to PKC. However, there are apparent differences between PKC and PfCDPK1 (e.g. the autophosphorylation activity of PfCDPK1 is affected in the presence of RKIP, whereas that of PKC remains unaffected), suggesting that the actual regulation mechanism may be different from that described for the Raf-1/RKIP/PKC context, which is an agreement with the finding that Plasmodium does not contain a classical three-component MAPK pathway [35]. Acknowledgements We thank Martin Gengenbacher and Christian M¨oskes for technical support and Drs. Sylke M¨uller and Petra Rohrbach for critical reading of the manuscript. The work was supported by funds of the Deutsche Forschungsgemeinschaft (DFG-project KA 1491/2-1) and includes parts of the doctoral thesis of Kirsten Pl¨uckhahn and Dominik Kugelstadt.

References [1] McCallum-Deighton N, Holder AA. The role of calcium in the invasion of human erythrocytes by Plasmodium falciparum. Mol Biochem Parasitol 1992;50:317–23. [2] Wasserman M, Alarcon C, Mendoza PM. Effects of Ca++ depletion on the asexual cell cycle of Plasmodium falciparum. Am J Trop Med Hyg 1982;31:711–7. [3] Tanabe K. Ion metabolism in malaria-infected erythrocytes. Blood Cells 1990;16:437–49. [4] Garcia CR. Calcium homeostasis and signaling in the blood-stage malaria parasite. Parasitol Today 1999;15:488–91. [5] Kirk K. Membrane transport in the malaria-infected erythrocyte. Physiol Rev 2001;81:495–537. [6] Billker O, Dechamps S, Tewari R, et al. Calcium and a calcium-dependent protein kinase regulate gamete formation and mosquito transmission in a malaria parasite. Cell 2004;117:503–14. [7] Kappes B, Doerig CD, Graeser R. An overview of Plasmodium protein kinases. Parasitol Today 1999;15:449–54. [8] Harper JF, Harmon A. Plants, symbiosis and parasites: a calcium signalling connection. Nat Rev Mol Cell Biol 2005;6:555–66. [9] Farber PM, Graeser R, Franklin RM, Kappes B. Molecular cloning and characterization of a second calcium-dependent protein kinase of Plasmodium falciparum. Mol Biochem Parasitol 1997;87:211–6. [10] Li JL, Baker DA, Cox LS. Sexual stage-specific expression of a third calcium-dependent protein kinase from Plasmodium falciparum. Biochim Biophys Acta 2000;1491:341–9. [11] Ward P, Equinet L, Packer J, Doerig C. Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote. BMC Genomics 2004;5:79. [12] Zhao Y, Kappes B, Franklin RM. Gene structure and expression of an unusual protein kinase from Plasmodium falciparum homologous at its carboxyl terminus with the EF hand calcium-binding proteins. J Biol Chem 1993;268:4347–54. [13] Ishino T, Orito Y, Chinzei Y, Yuda M. A calcium-dependent protein kinase regulates Plasmodium ookinete access to the midgut epithelial cell. Mol Microbiol 2006;59:1175–84. [14] Silva-Neto MA, Atella GC, Shahabuddin M. Inhibition of Ca2+ / calmodulin-dependent protein kinase blocks morphological differentiation of plasmodium gallinaceum zygotes to ookinetes. J Biol Chem 2002;277:14085–91. [15] Bernier I, Tresca JP, Jolles P. Ligand-binding studies with a 23 kDa protein purified from bovine brain cytosol. Biochim Biophys Acta 1986;871:19–23. [16] Frayne J, Ingram C, Love S, Hall L. Localisation of phosphatidylethanolamine-binding protein in the brain and other tissues of the rat. Cell Tissue Res 1999;298:415–23. [17] Serre L, Vallee B, Bureaud N, et al. Crystal structure of the phosphatidylethanolamine-binding protein from bovine brain: a novel structural class of phospholipid-binding proteins. Structure 1998;6:1255–65. [18] Bruun AW, Svendsen I, Sorensen SO, et al. A high-affinity inhibitor of yeast carboxypeptidase Y is encoded by TFS1 and shows homology to a family of lipid binding proteins. Biochemistry 1998;37:3351–7. [19] Chautard H, Jacquet M, Schoentgen F, et al. Tfs1p, a member of the PEBP family, inhibits the Ira2p but not the Ira1p Ras GTPase-activating protein in Saccharomyces cerevisiae. Eukaryot Cell 2004;3:459–70. [20] Pnueli L, Gutfinger T, Hareven D, et al. Tomato SP-interacting proteins define a conserved signaling system that regulates shoot architecture and flowering. Plant Cell 2001;13:2687–702. [21] Hengst U, Albrecht H, Hess D, Monard D. The phosphatidylethanolaminebinding protein is the prototype of a novel family of serine protease inhibitors. J Biol Chem 2001;276:535–40. [22] Yeung K, Seitz T, Li S, et al. Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP. Nature 1999;401:173–7. [23] Keller ET, Fu Z, Brennan M. The role of Raf kinase inhibitor protein (RKIP) in health and disease. Biochem Pharmacol 2004;68:1049–53. [24] Ojika K, Mitake S, Tohdoh N, et al. Hippocampal cholinergic neurostimulating peptides (HCNP). Prog Neurobiol 2000;60:37–83.

D. Kugelstadt et al. / Molecular & Biochemical Parasitology 151 (2007) 111–117 [25] Tsugu Y, Ojika K, Matsukawa N, et al. High levels of hippocampal cholinergic neurostimulating peptide (HCNP) in the CSF of some patients with Alzheimer’s disease. Eur J Neurol 1998;5:561–9. [26] Trager W, Jensen JB. Human malaria parasites in continuous culture. Science 1976;193:673–5. [27] Trottein F, Cowman AF. The primary structure of a putative phosphatidylethanolamine-binding protein from Plasmodium falciparum. Mol Biochem Parasitol 1995;70:235–9. [28] Zhao Y, Franklin RM, Kappes B. Plasmodium falciparum calciumdependent protein kinase phosphorylates proteins of the host erythrocytic membrane. Mol Biochem Parasitol 1994;66:329– 43. [29] Kinter M, Sherman NE. Protein sequencing and identification using tadem mass spectrometry. New York: Wiley; 2000. p. 147–65. [30] Le Roch KG, Zhou Y, Blair PL, et al. Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 2003;301: 1503–8.

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[31] Bozdech Z, Llinas M, Pulliam BL, et al. The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol 2003;1:E5. [32] Corbit KC, Trakul N, Eves EM, et al. Activation of Raf-1 signaling by protein kinase C through a mechanism involving Raf kinase inhibitory protein. J Biol Chem 2003;278:13061–8. [33] Moskes C, Burghaus PA, Wernli B, et al. Export of Plasmodium falciparum calcium-dependent protein kinase 1 to the parasitophorous vacuole is dependent on three N-terminal membrane anchor motifs. Mol Microbiol 2004;54:676–91. [34] Bucquoy S, Jolles P, Schoentgen F. Relationships between molecular interactions (nucleotides, lipids and proteins) and structural features of the bovine brain 21-kDa protein. Eur J Biochem 1994;225:1203–10. [35] Dorin D, Semblat JP, Poullet P, et al. PfPK7, an atypical MEK-related protein kinase, reflects the absence of classical three-component MAPK pathways in the human malaria parasite Plasmodium falciparum. Mol Microbiol 2005;55:184–96.