A MAP kinase homologue from the human malaria parasite, Plasmodium falciparum

GENE AN ~NTERNATIONAL ,JOURNAL ON GENES AND GENOME5

ELSEVIER

Gene 177 (1996) 1-6

A MAP kinase homologue from the human malaria parasite,

Plasmodiumfalciparum Caroline M. Doerig a, Daniel Parzy b, Gordon Langsley c, Paul Horrocks a, Richard Carter e, Christian D. Doerig a., a Unitd I N S E R M 399, Facultd de M~decine de Marseille, 27 Bd. Jean Moulin, 13385 Marseille Cddex 5, France b Institut de Mddecine Tropicale du Service de Santk des Armkes, Le Pharo, 13998 Marseille, France c Institut Pasteur, Experimental Parasitology, 25-28 Rue du Dr. Roux, 75724 Paris, France d Institute of Cell and Molecular Biology, University of Edinburgh, Mayfield Road, Edinburgh, EH9 3JR, UK e Institute of Cell, Animal and Population Biology, University of Edinburgh, West Mains Road, Edinburgh, EH9 3JT, UK

Received 12 February 1996; accepted 12 March 1996

Abstract Pfmap-1, a gene encoding a novel protein kinase, has been identified in the human malaria parasite Plasmodium falciparum, using the polymerase chain reaction with degenerate oligodeoxyribonucleotides designed to hybridise to conserved regions of cdc2-related kinases. Computer comparison with other protein kinases strongly suggests that the protein encoded by this gene is closely related to mitogen-activated protein (MAP) kinases, which play important roles in eukaryotic adaptative response and signal transduction. In addition to the conserved MAP kinase catalytic domain, Pfmap-1 contains a highly charged C-terminal extension that includes two sets of repeated amino acid motifs. Pfmap-1 is located on chromosome 14 of P. falciparum, and its mRNA has a size of 3.7 kb. Keywords: Mitogen-activated protein kinase; Parasitic protozoans; A, adenosine

1. Introduction

The emergence of drug-resistance in the malaria parasite Plasmodiumfalciparum represents a serious threat to tropical public health. The development of novel chemotherapeutic agents would be facilitated by a greater knowledge of the molecular mechanisms regulating parasite growth and differentiation. This prompted us to initiate a study aimed at identifying and analysing regulatory networks operating in P. falciparum. In this context we became interested in protein kinases, especially those related to the cdc2 family. Protein kinases are essential elements of the biochemical pathways regulating cell growth, development and differentiation. The cdc2 gene of Schizosaccharomyces * Corresponding author. Tel. + 33 91 834523;Fax + 33 91 796063. Abbreviations: aa, amino acid(s); bp, base pair(s); kb, kilobase(s) or 1000bp; nt, nucleotides;MAP kinase, mitogen-activatedprotein kinase; ORF, open reading frame; PCR, polymerasechain reaction;Pfmap-1, P. falciparum mitogen-activated protein kinase 1; Pfcrk-1, P. falciparum cdc2-relatedkinase 1; PfPk5, P.falciparum proteinkinase 5; T, thymidine. 0378-1119/96/$15.00© 1996 ElsevierScienceB.V. All rights reserved PH S0378-1119(96)00281-8

pombe (like its homologue in Saccharomyces cerevisiae,

CDC28) encodes a 34-kDa protein kinase that controls both entry into mitosis and G1/S transition (Beach et al., 1982, reviewed in Forsburg and Nurse, 1991). The activity of this kinase is modulated by its own state of phosphorylation and association with positive (cyclins) or negative (ckis) regulatory proteins (reviewed in Peter and Herskowitz, 1994). Homologues of the cdc2 gene have been identified in numerous eukaryotes (e.g. Lehner and O'Farrell, 1990; Feiler and Jacobs, 1990; RossMcDonald et al., 1994). Mitogen-activated protein (MAP) kinases, also called ERK (extracellularly-regulated kinases), form a family of serine/threonine kinases with homology to the cdc2 family. They function at one of the last kinase steps in signal transduction pathways triggered by a wide range of stimuli such as hormone treatment or high osmolarity (reviewed in Neiman, 1993). Their activity is regulated by their own state of phosphorylation (Payne et al,, 1991; Posada and Cooper, 1992; Seger et al., 1991). M A P kinase substrates include cell cycle control elements and transcription factors responsible for expression of genes involved in adaptative

2

CM. Doerig et al./Gene 177 (1996) 1 6

response. For example, mating in Saccharomyces cerevisiae is initiated by a pheromone/receptor interaction that transmits a signal via G proteins to the Stell kinase (a M A P K K K , or MAP kinase kinase kinase). Stell then phosphorylates the MAP kinase kinase Ste7, which in turn phosphorylates the MAP kinases Fus3 and KSS1. Once activated, these MAP kinases phosphorylate substrates involved in mating, such as a CDC28 inhibitor (which results in cell cycle arrest) and the Stel2 transcription factor, which activates the transcription of genes involved in differentiation (reviewed in Bardwell et al., 1994; Errede and Levin, 1993; Oehlen and Cross, 1994). Here we describe a P. falciparum gene, Pfmap-1, that was found during a search for cdc2-related kinases in this organism. Pfmap-I presents all the features required to classify it as a member of the MAP kinase gene family. The putative protein encoded by Pfmap-1 shows a relatively high level of homology with Spkl, a S. pombe homologue of the KSS1/Fus3 kinases involved in S. cerevisiae mating.

2. Experimental and discussion 2.1. Isolation of a cDNA clone encoding a novel protein kinase

clones were isolated from a gametocyte cDNA library, using the C2 insert as a probe. One of these contained an overlap of approximately 800 bp with the 5' end of the C2 insert, plus additional upstream sequences. This allowed the identification of two in-frame ATGs that may function as start codons (positions 100-102 and 127-129); because the former is embedded in A and T homopolymeric stretches which are characteristic of P. falciparum untranslated sequences, and because the latter encodes a methionine followed by proline and lysine residues (the three residues found at the amino-terminus of a yeast MAP kinase, see Fig. 2), we favor the hypothesis that the most downstream of the two ATGs is the functional start codon, although this remains to be determined experimentally. An in-flame stop codon, surrounded by sequences that are rich in A and T homopolymers, is present 30 bp upstream of the first in-flame ATG (nt 67-69), confirming that translation is likely to initiate at one of these two ATGs. As none of the additional clones extended further downstream than the C2 insert, we employed the inverse PCR method (Triglia et al., 1988), which enabled us to identify a stop codon at nt 2605-2607 (see legend to Fig. 1). The polypeptide potentially encoded by the O R F is 826 amino acids long, with a predicted molecular mass of 96 460 Da.

2.2. Pfmap-I is related to the MAP kinase gene family Our purpose was to identify P.falciparum genes encoding cdc2-related protein kinases. Using degenerate primers 1 and 2 (see Fig. 1) designed to hybridise to conserved regions of the p34 cat2 family, we obtained a PCR product of the expected size (approximately 500 bp) from a P. falciparum clone 3D7 (Walliker et al., 1987) gametocyte cDNA library. Southern hybridisation studies using this product as a probe suggested that the 500-bp band was in fact a mixture of several different species of molecules (data not shown). Therefore the PCR products were cloned into the pCRII vector (Invitrogen), and individual clones sequenced. This allowed the identification of several different inserts amplified from different protein kinase genes, including the previously described PfPK5, a P. falciparum putative cdc2 homologue (RossMcDonald et al., 1994), and Pfcrk-1, a novel P. falciparum gene encoding a developmentally regulated cdc2-related protein kinase (Doerig et al., 1995). One clone (hereafter called C1) carried a 516-bp insert containing an uninterrupted open reading frame (ORF) not previously characterised from P.falciparum, with homology to a variety of cdc2- and MAP-related kinases. The C1 insert was used as a probe to screen a P. falciparum 3D7 gametocyte cDNA library constructed in the pJFEDAF vector (Elliot et al., 1990). A cDNA clone with a 1.9-kb insert (clone C2) was isolated and shown to contain an O R F spanning its entire length (see legend to Fig. 1). To define the start and stop codons of the putative protein encoded by the C 2 0 R F , additional

A computer analysis using the M O T I F S algorithm (Devereux et al., 1984) confirmed that the O R F encodes a protein kinase, as both protein kinase signatures (Bairoch, 1988) are present: the sequence V G K G A Y G V V (aa 29-37), which corresponds to the ATP-binding domain of protein kinases, and the sequence L L H R D I K P S N I L V (aa 142-154), located in the central part of the catalytic domain of serine/threonine protein kinases. Furthermore, the O R F contains the 15 invariant amino acids characteristic of serine/threonine protein kinases (Hanks et al., 1988; Hanks and Quinn, 1991) (Fig. 2). TFASTA analysis (Devereux et al., 1984) indicated that the polypeptide potentially encoded by the ORF is a member of the MAP kinase family: most of the 60 highest score entries were MAP kinases. The two residues whose phosphorylation is required for activation of all MAP kinases (Payne et al., 1991; Posada and Cooper, 1992; Seger et al., 1991) are found in the O R F (Fig. 2); only one of these residues is found in cdc2 family members. The catalytic domain of the putative kinase encoded by the ORF is 47% identical to the Spkl (Toda et al., 1990) catalytic domain and 41-44% identical to that of other MAP kinases. Homology to members of the cdc2 family is lower, with 38%, 35% and 37% identity to PfPK5, Pfcrk-1 and S. pombe cdc2 respectively. Moreover, the ORF does not encode a PSTAIRE-like motif characteristic of members of the cdc2 family. On the basis of these

CM. Doerig et al./Gene 177 (1996) 1-6 TT AT CAATTTAAAAC M ~

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Fig. 1. Sequence of Pfmap-1. The deduced amino acid sequence is indicated above the nucleotide sequence. The location of PCR primers is indicated by arrows. Start and stop codons discussed in Section 2.1 are in underlined bold characters, and the amino acid repeats discussed in Section 2.2 are underlined. The boundaries of the C2 insert are indicated by A symbols. Methods. The degenerate primers used to amplify cdc2-related protein kinase gene fragments, and hence marking the boundaries of clone C1, were: primer 1, GAAAAAAT(A/T)GG(A/T)GAAGG(A/T)AC(A/T)TA and primer 2, ACTTC(A/T)GG(A/T)GCT CT(A/G)TACCA. PCR conditions with these primers were as previously described (Doerig et al., 1995). The stretch of As indicated in bold characters between positions 933 and 944 contained 13 residues in the C2 cDNA insert; this would result in a frameshift. This region was amplified with primers 3 and 4 (see the figure) from P. falciparum 3D7 genomic DNA and sequenced. There were 12 A residues in the polyadenosine stretch amplified from genomic DNA, suggesting that a frameshift mutation had occurred during the cDNA cloning (other cDNA clones did not contain this frameshift mutation). Sequences upstream of the C2 insert were determined from an overlapping cDNA clone, and sequences downstream of the C2 insert were determined by inverse PCR (Triglia et al., 1988) with primers invl and inv2, using as a template genomic 3D7 DNA that had been cleaved with MboI1 and religated. Sequences beyond the downstream MbolI site were obtained by direct sequencing of PCR products from a cDNA library using primer 5 and a primer in the vector sequence. The nucleotide sequence is available in the EMBL, GenBank and DDBJ databases under the accession No. X82646.

4

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Fig. 2. Amino acid comparison of the catalytic domain of Pfmap-1 with that of other MAP kinases [S, pombe Spkl (Toda et al., 1991); S. cerevisiae FUS3 (Elion et al., 1990) and KSS1 (Courchesne et al., 1989); Mus musculusERK2 (Her et al., 1991); Drosophila melanogaster ERK-A (Briggs and Zipuski, 1992); Arabidopsis thaliana ATMPK (Mizoguchi et al,, 1993)] (between 4 and 11 residues from the carboxy end of these kinases do not appear on the figure). Dots (.) indicate identity, dashes (-) indicate gaps introduced to optimise alignment. The figure is derived from an alignment performed using the GeneWorks software. The 15 invariant residues of serine/threonine kinases are labelled with an (©). The two protein kinase signatures are indicated in bold characters. The (x) symbols locate the two residues (Thr-184 and Tyr-186) that correspond to regulatory phosphorylation sites in members of the MAP kinase family. The level of homology is expressed as the percentage of identical amino acids, within the catalytic domain, in Pfmap-1 and each MAP kinase mentioned. The boundaries used to define the catalytic domain for this analysis are indicated by vertical dashes (I). observations, we propose that this O R F encodes a P.

(alciparum M A P kinase h o m o l o g u e , hereafter called Pfmap-1. In addition to the kinase catalytic domain, Pfmap-1 contains a C-terminal extension of 503 amino acids that is rich in basic and acidic residues. A prominent feature of this extension is the presence of 5 repeats of the tetramer I K E Q (aa 535-554) and of 13 imperfect repeats of the octamer K K Y V D ( G / S / E ) ( G / S / L ) N (aa 672-775). Such repeats are not u n c o m m o n in P. falciparum proteins (Anders et al., 1988). Pfcrk-1 contains a similar highly charged region with repeated motifs at its N-terminus (Doerig et al., 1995). The function of these elements remains unknown.

c h r o m o s o m e s were separated by pulse-field gradient electrophoresis, transferred to a nylon filter and hybridised to a Pfmap-1 probe. This showed that Pfmap-1 is located on c h r o m o s o m e No. 14 (Fig. 3B). The location was confirmed by hybridisation of the filter to an aldolase probe; the P. falciparum aldolase gene is k n o w n to reside on c h r o m o s o m e 14 (Triglia et al., 1992). A Northern blot performed with total R N A from asexual parasites is presented in Fig. 3C. This confirmed that Pfmap-1 is transcribed during infection of the erythrocyte, and indicated that the Pfmap-1 m R N A has a size of approximately 3.7 kb. Although this is 1.3 kb longer than the ORF, it is line with results from other P. falciparum genes (Levitt et al., 1993).

2.3. Chromosomal mapping and transcript size of Pfmap-1 3. Conclusions

P.falciparum genomic D N A was digested with a panel of restriction enzymes and hybridised with a Pfmap-1 probe (Fig. 3A). The presence of positive hybridisation signals confirmed that Pfmap-I is a P. falciparum gene. In each case, a single band was observed, which is consistent with Pfmap-1 being represented in the P. falciparum genome as a single copy gene. P. falciparum

Pfmap-1, a gene encoding a putative M A P kinase homologue, has been identified in P.falciparum, suggesting that signal transduction networks similar to those found in other eukaryotes may operate in this organism. Pfmap-1 is located on c h r o m o s o m e 14, presumably as a single copy; the Pfmap-1 m R N A detected in asexual

C.M. Doeriget al./Gene 177 (1996) 1 6

5

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Fig. 3. Hybridisation experiments. Hybridisation conditions were as described (Doerig et al., 1995). (A) Southern blot. Approximately 1/~g of genomic DNA from P. falciparum clone 3D7 was digested with restriction enzymes, transferred to a Hybond N + membrane (Amersham) and hybridised to a Pfmap-1 probe. The probe was a 32p-labelled PCR product amplified from clone C2 with primer 1 and 2 (see Fig. 1). Restriction enzymes were (lanes 1-12): AccI, BgllI, Sau3A, MspI, XhoI, DraI, HinclI,HindlII, AluI, Hinfl, XbaI and NheI. The sizes of comigrating molecular mass markers are indicated to the left in kb. (B) Chromosomal mapping. Chromosomes from P.falciparum K1 isolate (Thaitong and Beale, 1981) were separated by pulse field gradient gel electrophoresis as described previously (Kemp et al., 1985), depurinated and transferred to a Hybond N + membrane. The membrane was probed with the same probe as in A (lane 2), or with a P. falciparum aldolase probe (lane 3). Lane 1 is an ethidium bromide stain of the chromosome gel prior to transfer. (C) Northern blot. 10/zg of total RNA from asexual parasites (K1 P. falciparum isolate) were used for the analysis, which was performed as previously described (Doerig et al., 1995). The probe was a PCR product covering the Pfmap-1 ORF from nt 126 to 2023.

parasites is approximately 3.7 kb long. In addition to the catalytic domain, the gene encodes a large, highly charged C-terminal extension containing two sets of repeated motifs. The function of this gene in the P. falciparum life cycle remains to be determined. By analogy with the functions of MAP kinases in other organisms, Pfmap-1 may be involved in the control of growth and/or sexual differentiation. The relatively high level of homology of Pfmap-I with Spkl, a S. pombe MAP kinase that is activated during response to mating pheromone, lends support to this hypothesis.

Acknowledgement

We thank P. Alano, A. Craig and D. Kaslow for kindly providing cDNA libraries. We were able to use DNA blots prepared by D. Arnot and S. Cheesman. P a r t s o f the s e q u e n c e w e r e v e r i f i e d o n a u t o m a t e d s e q u e n c e r s w i t h t h e h e l p o f C. J o b l e t a n d M . O n a t e (Assistance Publique de Marseille, Programme H o s p i t a l i e r de R e c h e r c h e C l i n i q u e 1995) a n d o f A l a i n R i c o (I.M.T.S.S.A.). T h e Pfmap-1 g e n e was i n d e p e n d e n t l y i s o l a t e d by R. G r a e s e r a n d B. K a p p e s at the

B i o z e n t r u m , U n i v e r s i t y o f Basel, S w i t z e r l a n d , w h o s e w o r k is to be p u b l i s h e d elsewhere. W e t h a n k t h e s e c o l l e a g u e s for freely d i s c u s s i n g the d a t a b e f o r e p u b l i c a tion. T h i s w o r k w a s s u p p o r t e d by a n M R C g r a n t to R.C. a n d b y t h e I n s t i t u t N a t i o n a l de la Sant6 et de la R e c h e r c h e M 6 d i c a l e ( I N S E R M ) U n i t 399.

References

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6

C.M. Doerig et al./Gene 177 (1996) 1 6

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