Identification of mitogen-activated protein kinase homologues from Leishmania mexicana

International Journal for Parasitology 33 (2003) 1577–1587 www.parasitology-online.com

Identification of mitogen-activated protein kinase homologues from Leishmania mexicanaq Martin Wiesea,*, Qiong Wangb, Iris Go¨rckeb a

Bernhard-Nocht-Institute for Tropical Medicine, Parasitology Section, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Germany b Max-Planck-Institut fu¨r Biologie, Abteilung Membranbiochemie, Corrensstrasse 38, D-72074 Tu¨bingen, Germany Received 21 July 2003; received in revised form 13 August 2003; accepted 18 August 2003

Abstract Mitogen-activated protein kinases are key-regulatory elements in the differentiation, proliferation, apoptosis and stress response of eukaryotic cells. Our recent identification of a mitogen-activated protein kinase homologue in Leishmania mexicana which is essential for the proliferation of the amastigote stage of the parasite living in the parasitophorous vacuole of the infected macrophage prompted us to screen the genome of L. mexicana for additional mitogen-activated protein kinase homologues using degenerate oligonucleotide primers in a polymerase chain reaction amplification approach. We cloned and sequenced the genes for eight new mitogen-activated protein kinase homologues which were subsequently shown to be present in one copy per haploid genome. The mRNA levels of the kinases varied significantly in pro- and amastigote life stages of the parasite. We used the structural information of the p38 stress-activated protein kinase, which belongs to the family of mitogen-activated protein kinases, for the alignment of the deduced proteins and the verification of the predicted secondary structure elements. All new mitogen-activated protein kinases reveal the typical 12 subdomain primary structure, the conserved residues characterising serine/threonine protein kinases and the characteristic TXY motif in the phosphorylation lip. Typical features of some of the molecules are amino acid insertions between the subdomains and long carboxy-terminal amino acid extensions carrying putative src-homology 3-binding motifs. q 2003 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Mitogen-activated protein kinase; Signal transduction; mRNA abundance; Protozoan parasite; Gene cloning; Sequence alignment

1. Introduction Mitogen-activated protein (MAP) kinases are keyregulatory elements in differentiation, proliferation, apoptosis and stress responses. In higher eukaryotic cells they are activated by dual phosphorylation on threonine and tyrosine residues of the conserved TXY motif in the phosphorylation lip. This leads to a conformational change in the molecule and an increase of kinase activity by about 3000 fold. Phosphorylation of the MAP kinase is brought about by a highly specific MAP kinase kinase, which in turn is activated by phosphorylation through a MAP kinase kinase kinase. These three molecules comprise the core module of q Nucleotide sequences reported in this paper are available in the GenBank database under accession numbers . .AJ293280–AJ293288. * Corresponding author. Tel.: þ 49-40-42818-498; fax: þ 49-4042818-400. E-mail address: [email protected] (M. Wiese).

the MAP kinase signal transduction cascade. In the yeast Saccharomyces cerevisiae five such modules have been described. They regulate mating via the MAP kinase FUS3, the response to changes in osmolarity via HOG1 and MPK1 and the response to starvation leading to filamentation and invasive growth in the case of KSS1 or sporulation in the case of SMK1 (Schaeffer and Weber, 1999). In the genome of Caenorhabditis elegans 14 MAP kinase family members have been identified (Plowman et al., 1999). Twenty-two proteins belonging to the MAP kinase family are described in mammalian cells to date (Pearson et al., 2001). For most of these MAP kinases a number of substrates and their activating upstream components have been identified (Garrington and Johnson, 1999). Most MAP kinases consist of small amino- and carboxy-terminal regions, the eukaryotic kinase domain which spans about 300 amino acids and can be divided into 12 subdomains that fold into a common catalytic core structure and a characteristic amino acid insertion between subdomains X and XI (Hanks and Hunter,

0020-7519/$30.00 q 2003 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S0020-7519(03)00252-2

1578

M. Wiese et al. / International Journal for Parasitology 33 (2003) 1577–1587

1995). Some, like the extracellular signal-regulated kinases ERK5 and ERK7, do have large additional domains as their carboxy-terminal regions (Zhou et al., 1995), which in the case of ERK7 is known to regulate its activity and localisation (Abe et al., 1999). Three-dimensional structures have been solved for four unphosphorylated, inactive forms of MAP kinases, rat ERK2 (Zhang et al., 1994), human p38a (Wilson et al., 1996), murine p38 (Wang et al., 1997) and JNK3 (Xie et al., 1998). Moreover, the structures of phosphorylated active ERK2 (Canagarajah et al., 1997) and p38g (Bellon et al., 1999), and of p38 complexed with a highly specific pyridinyl-imidazole inhibitor (Tong et al., 1997; Wilson et al., 1997) have been described recently. Upon phosphorylation ERK2 forms dimers which promote nuclear localisation and might also influence substrate interactions (Cobb and Goldsmith, 2000). However, doubly phosphorylated active p38g exists as a monomer (Bellon et al., 1999). The overall three-dimensional structure of MAP kinases is two-lobed. The amino-terminal smaller lobe (subdomains I– IV) is primarily involved in anchoring and orienting the nucleotide substrate (ATP or GTP). It consists predominantly of an antiparallel b-sheet structure. By contrast, the larger carboxy-terminal lobe (subdomains VI – XI) is mainly a-helical. It is responsible for binding the peptide substrate and initiating the phosphate transfer. The two lobes are connected via subdomain V residues. The deep cleft between the lobes is the site of catalysis. The great progress that has been made in connecting the steps of many signal transduction pathways in higher eukaryotes was possible because of the availability of both a large number of cloned genes and specific antibodies leading to the identification of intermolecular interactions. MAP kinase-specific inhibitors have been identified which interfere with essential regulatory processes in the cells (Lee et al., 1994; Tong et al., 1997; Wilson et al., 1997). They comprise potential therapeutics against diseases, like cancer, caused by deregulation of differentiation and proliferation (Laird et al., 2000). On the other hand, diseases caused by eukaryotic pathogens like protozoans (e.g. Plasmodium falciparum, Leishmania species, Trypanosoma species, Toxoplasma gondii, Entamoeba histolytica) and helminths could in principle be treated by interruption of signal transduction cascades in the pathogen stopping them from proliferation and/or differentiation. In Leishmania mexicana, for instance, a suitable drug target has already

been identified – a MAP kinase homologue has been shown by deletion analysis to be essential for the proliferation of the amastigote stage of the parasite living in the parasitophorous vacuole of the infected macrophage (Wiese, 1998). Besides their potential medical importance MAP kinases in kinetoplastid protozoans are likely to be modulators of gene regulatory proteins like similar kinases are in higher eukaryotes. However, no transcription factors have been identified in trypanosomatids to date. It is generally anticipated that gene regulation occurs posttranscriptionally at the level of RNA processing or turnover, or at the level of translation and protein stability (Parsons and Ruben, 2000). Characterisation of MAP kinases from Leishmania may ultimately lead to the identification of their substrates and therefore to the elucidation of gene regulation in these organisms. Here we describe the cloning and characterisation of eight new MAP kinase genes from L. mexicana.

2. Materials and methods 2.1. Parasites Promastigotes of L. mexicana strain MNYC/BZ/ 62/M379 were grown in vitro as described previously (Menz et al., 1991). Lesion-derived amastigotes and axenic amastigotes were obtained as described previously (Wiese, 1998). 2.2. New genes and DDBJ/EMBL/GenBank accession numbers LmxMPK2, AJ293280; LmxMPK3, AJ293281; LmxMPK4, AJ293282; LmxMPK5, AJ293283; LmxMPK6, AJ293284; LmxMPK7, AJ293285; LmxMPK8, AJ293286; LmxMPK9, AJ293287; LmxCRK, AJ293288. 2.3. Oligonucleotides Oligonucleotides for genomic polymerase chain reaction (PCR) amplification (Table 1) and sequencing were purchased from Interactiva The Virtual Laboratory (Ulm, Germany).

Table 1 Oligonucleotides used for cloning of the mitogen-activated protein kinase homologues from L. mexicana Primer

Sequence (50 to 30 )

Corresponding peptide sequence

HRD2 HRD3 ATRW1 ATRW2 VTRW1 VTRW2

50 -CA(CT)CG(GCAT)GA(CT)(GCAT)T(GCAT)AA(GA)CC-30 50 -CA(CT)AG( C T)GA(CT)(GCAT)T(GCAT)AA(GA)CC-30 50 -C(GT)(GA)TACCA( C T)CT(GCAT)GT(GCAT)GC-30 50 -C(GT)(GA)TACCA(GCAT)CG(GCAT)GT(GCAT)GC-30 50 -C(GT)(GA)TACCA( C T)CT(GCAT)GT(GCAT)AC-30 50 -C(GT)(GA)TACCA(GCAT)CG(GCAT)GT(GCAT)AC-30

HRD(VLMFI)KP HRD(VLMFI)KP ATRWY(RS) ATRWY(RS) VTRWY(RS) VTRWY(RS)

M. Wiese et al. / International Journal for Parasitology 33 (2003) 1577–1587

2.4. Genomic PCR, gene cloning, and reverse transcriptase-PCR Total DNA from L. mexicana promastigotes was isolated as described previously (Medina-Acosta and Cross, 1993) and used as a template to perform a touch-down PCR with the Expande High Fidelity PCR System (Roche Molecular Biochemicals, Mannheim, Germany). Degenerate oligonucleotide sense and anti-sense primers were used at a concentration of 0.6 mM on 30 ng of DNA and 0.2 mM dNTPs in PCR reaction buffer þ Mg2þ provided by the manufacturer and 0.75 units High Fidelity Polymerase in a total volume of 50 ml. Starting with 5 min at 95 8C, 35 cycles were performed as follows: 1 min at 94 8C, 30 s at 55 8C (this temperature was consecutively reduced by 1 8C/cycle down to 40 8C in the first 15 cycles and then kept at 40 8C for another 20 cycles), 30 s at 72 8C. Finally, the reaction mixture was kept at 72 8C for another 7 min. PCR products were analysed on agarose gels, positives subcloned into pCR2.1 using the TA-cloning kit (Invitrogen, San Diego, CA) and sequenced. Those fragments carrying parts of putative kinase genes were isolated, digoxigenin (DIG)-labelled and used to screen a genomic DNA-library of L. mexicana (Wiese et al., 1995). Positive phage clones were isolated and their inserts subcloned into pBluescript SKII(þ ) (Stratagene, La Jolla, CA). Consecutive sequencing was performed starting with the known sequence of the original PCR fragment towards both ends of the respective open reading frames using the Autoreade Sequencing Kit (Amersham-Pharmacia, Freiburg, Germany) and an ALFexpress sequencing device (Amersham-Pharmacia). Both strands were sequenced. The GCG/Wisconsin software (Devereux et al., 1984) was used to assemble and analyse the DNA sequences. Determination of splice addition sites by reverse transcriptase-PCR (RT-PCR) followed by a second nested PCR, cloning and sequencing was performed as described before using the mini-exon primer and two gene internal primers (Wiese, 1998). 2.5. Southern hybridisation Total cellular DNA (3 mg) was hydrolysed with restriction endonucleases and the products were separated by electrophoresis in 0.7% agarose gels. The DNA was transferred to Biodyne A nylon membranes (Pall, Dreieich, Germany), UV-crosslinked and subjected to hybridisation with DIGlabelled probes and detection of chemiluminescence following the instructions of the manufacturers (Roche Molecular Biochemicals, Mannheim, Germany). The membranes were stripped several times and reused for the different probes.

1579

amastigotes) and amastigotes isolated from lesions of Balb/c mice were used to isolate total RNA and DNA. Cells (5 £ 108) were resuspended in 1 ml of Trizol (Invitrogen) and lysed by four freeze – thaw cycles with alternating exposure to liquid nitrogen and 37 8C in a water bath. Then total RNA and DNA were obtained according to the manufacturer’s protocol. The RNA was dissolved in 540 ml diethyl pyrocarbonate-treated ddH2O, 50% deionised formamide, and 7% formaldehyde and denatured by incubation at 68 8C for 15 min. Finally, 2 volumes of 20 £ SSC were added to make up a final volume of 1.6 ml. The DNA was resuspended in 1.6 ml ddH2O, denatured by boiling for 10 min, immediately followed by 5 min on ice. Either DNA or RNA solution (80 ml) was spotted on Biodyne A (Pall) and Roche positively-charged nylon membranes, respectively, using a dot blot manifold (SRC 96 D Minifold, Schleicher and Schuell, Dassel, Germany). Nucleic acids were UV-crosslinked to the membranes, prehybridised in DIG Easy Hyb solution (Roche) containing 100 mg/ml of fish sperm DNA, and hybridised to DIGlabelled DNA probes according to the manufacturer’s protocol (Roche). Low- and high-stringency washes were performed as recommended at 62 and 50 8C for DNA and RNA, respectively. Hybridisation was detected using the DIG-detection kit (Roche) and the membranes were exposed to X-ray films. The films were scanned and the intensity of the dots was measured using Image J software (http://rsb.info.nih.gov/ij/). 2.7. Analysis of sequence data Initial alignments of amino acid sequences were performed using the PILEUP program of the GCG/Wisconsin sequence analysis package (Devereux et al., 1984) followed by manual modification. To increase the accuracy long carboxy-terminal extensions were omitted from the alignment. The sequence and structure of p38 (Wang et al., 1997) were used to prevent the introduction of gaps into secondary structure elements which were predicted using the JPred2 sequence analysis server with the JNet secondary structure prediction algorithm (Cuff et al., 1998; Cuff and Barton, 2000). Searches of public domain protein sequence databases were carried out with BLAST (Altschul et al., 1997) through the NCBI web server (http://www.ncbi.nlm. nih.gov) and TFASTA of the GCG/Wisconsin sequence analysis package using the default options.

3. Results 3.1. Cloning of MAP kinase homologues from L. mexicana

2.6. Isolation and quantification of total RNA and DNA from Leishmania Promastigotes from logarithmic and stationary growth phase, in vitro differentiated amastigotes (axenic

Mitogen- and stress-activated protein kinases contain characteristic amino acid sequences (Ku¨ltz, 1998). In particular, residues found in subdomain VIb and subdomain VIII are highly conserved (Fig. 1). Therefore, we chose

1580

M. Wiese et al. / International Journal for Parasitology 33 (2003) 1577–1587

Fig. 1. Typical mitogen-activated protein kinase catalytic domain. The 12 conserved subdomains are indicated by Roman numerals. Consensus sequences found in the subdomains are shown.

the amino acid stretches HRDXKP and BTRWYZ (X is either valine, leucine, methionine, phenylalanine or isoleucine; B is alanine or valine; Z is arginine or serine) for the generation of degenerate oligonucleotide primers designed for the amplification of gene fragments corresponding to the phosphorylation lip of protein kinases in a PCR from genomic DNA of L. mexicana (Table 1). Amplified fragments were subcloned, sequenced and assigned to be positive on the occurrence of the DFG amino acid motif indicative for protein kinases. Forty-eight positive clones were found after subcloning the PCR fragments obtained by the eight possible combinations of the oligonucleotide primers. LmxMPK1 (designated lmpk in Wiese, 1998) was represented 20 times, however, eight new potential kinase gene fragments varying in size from 131 to 338 bp were identified (Table 2). One of these fragments revealed homology to cdc2-related kinases displaying one serine residue as a single potential phosphorylation site in the SHE-motif of its T-loop, a structure analogous to the phosphorylation lip present in MAP kinases (Morgan, 1995). All other fragments contained the dual phosphorylation motif TXY indicative of MAP kinases close to the end of the potential phosphorylation loop in subdomain VIII.

The fragments were DIG-labelled and used as probes to screen a genomic DNA library of L. mexicana (Wiese et al., 1995). Inserts of positive phage clones were subcloned and used for sequencing. Inspection of the sequences generated in the Leishmania genome project led to the identification of one additional open reading frame on chromosome 19 of Leishmania major Friedlin with high homology to MAP kinases (accession number AL139796). Using L. major gene-specific oligonucleotides we amplified the corresponding gene from L. mexicana genomic DNA and used the labelled DNA to isolate the gene from the L. mexicana genomic DNA library. Full length sequences of all genes have been deposited in the DDBJ/EMBL/GenBank databases. Southern analysis using gene-specific DNA fragments as probes revealed that the nine MAP kinase homologues and the cdc2-related kinase are present as single copy genes per haploid genome of L. mexicana (Fig. 2). 3.2. mRNA abundance in different life stages Total RNA and DNA were extracted from the same samples of 5 £ 108 cells of logarithmic (3.4 £ 107 cells/ml)

Table 2 Characterisation of the cloned kinase genes and deduced amino acid sequences Kinase

Length of open reading frame [bp]

Length of deduced protein [aa]

Calculated molecular weight [kDa]

Phosphorylation site motifa

Insertions at position – lengtha

COOH-terminal extensionb

CD domain-like sequencea

LmxMPK1 LmxMPK2 LmxMPK3 LmxMPK4 LmxMPK5 LmxMPK6 LmxMPK7

1074 1374 1164 1089 1158 3318 1653

358 458 388 363 386 1106 551

41.0 50.5 43.7 41.5 43.9 118.9 61.8

TDY TDY TDY TQY TDY TDY TDY

– þþ þ – – þþ þ þ

PLVDE RPDDE DEEDE DAAEE DLGFD DGFRD DNRDE

LmxMPK8

4737

1579

165.7

TNY

þþ þ

–

LmxMPK9 LmxCRK p38

1221 1407 1080

407 469 360

45.0 52.2 41.3

TEY SHE TEY

– – – – – – D44 – 69 aa R250 – 42 aa E376 – 22 aa R150 – 62 aa G297 – 33 aa –

þþ

DSDDEc

–

–

DPDDE

a

Single letter code for amino acid residues. Length of carboxy-terminal extension: þþ þ , .700 amino acid residues; þþ , .70 amino acid residues; þ, .20 amino acid residues as compared to the shortest sequences (LmxMPK4 and LmxMPK5). c Not shown in Fig. 4, because it is located close to the end of the protein. b

M. Wiese et al. / International Journal for Parasitology 33 (2003) 1577–1587

1581

Fig. 2. Southern analysis of genomic DNA. DNA of L. mexicana cut with HincII (1), HindIII (2), or Acc65I (3) was electrophoresed on a 0.7% agarose gel, blotted onto nylon membranes and probed with digoxigenin-labelled DNA probes derived from the genes indicated below the blot. MPK2–MPK9, probes derived from LmxMPK2–LmxMPK9; CRK, probe derived from LmxCRK. Numbers indicate the approximate size of DNA markers in kb.

and stationary phase promastigotes (6.3 £ 107 cells/ml), axenic amastigotes (4.4 £ 107 cells/ml) and lesion-derived amastigotes. Fig. 3A shows the dot blots of these RNAs and DNAs hybridised to the DIG-labelled DNA probes specific for the nine MAP kinase homologues cloned from L. mexicana. As a non-kinase control a probe specific for the gene of the membrane-bound acid phosphatase (LmxMBAP) was included (Wiese et al., 1996). Moreover, DNA and RNA were isolated from the null mutant promastigotes for LmxMPK1 to serve as a negative control in the hybridisation to the LmxMPK1 probe. The presence of nucleic acids in this preparation was confirmed by hybridisation to the LmxMPK5 probe. The lowest hybridisation of total DNA to the different probes was observed for the lesion-derived amastigotes reflecting the difficulties in determination of the cell number due to cellular debris in this preparation. For the axenic amastigotes the concentration of cells/ml was also overestimated leading to a total cell number of less than 5 £ 108 in the nucleic acid preparation and therefore a weaker DNA-DNA-hybridisation signal with the DIG-labelled probes. The preparation of DNA from the two promastigote stages indicates roughly the same cell numbers. A quantitative analysis using the Image J software followed by normalisation of the RNA hybridisation to the amounts of DNA isolated from the same cells is shown in Fig. 3B. mRNAs for all genes analysed were found in the hybridisation albeit to varying amounts. Generally, axenic amastigotes showed relatively high mRNA levels for all genes. LmxMBAP is known to be present in the lysosomal compartments of promastigotes and amastigotes (Wiese et al., 1996) which correlates to the observed formation of mRNA in these life stages. LmxMPK1 has also been shown to be present in pro- and

amastigotes and was found to be absolutely required for the survival of the amastigote stage (Wiese, 1998). LmxMPK3 is the only gene which shows a tight down-regulation of the amount of mRNA in the lesion amastigote and high levels in promastigotes. The relatively high LmxMPK3 mRNA level in the axenic amastigotes might reflect the fact that these cells still display some promastigote characteristics. The amounts of mRNA for LmxMPK4 and LmxMPK5 are also relatively low in the lesion amastigotes showing a higher steady state level in the promastigotes. For LmxMPK5 the logarithmic phase promastigotes revealed the highest mRNA level. LmxMPK1 – LmxMPK4 showed about equal mRNA levels in the promastigote stages, whereas there was an up-regulation of mRNAs in the stationary phase promastigotes for LmxMPK6, LmxMPK8, and LmxMPK9. Finally, LmxMPK7 showed up-regulation of the mRNA level in the amastigotes. 3.3. Characterisation of the L. mexicana MAP kinase homologues The open reading frames of the MAP kinases varied in length from 1074 bp for LmxMPK1 to 4737 bp for LmxMPK8 corresponding to proteins in size ranging from 41 to 165.7 kDa (Table 2). Four different phosphorylation site motifs carrying D, Q, N or E as a central residue in the TXY motif were found. Fig. 4 shows the amino acid sequence alignment of the kinase domains of the nine MAP kinase homologues from L. mexicana using the stressactivated protein kinase p38 as the source for secondary structure information (Wang et al., 1997). The alignment was generated by a combination of different methods. First, secondary structures of all sequences including p38 were

1582

M. Wiese et al. / International Journal for Parasitology 33 (2003) 1577–1587

Fig. 3. Analysis of mRNA abundance of the mitogen-activated protein kinase homologues in different life stages of L. mexicana. (A) Dot blot of total RNA (top) and DNA (bottom) from L. mexicana wild type logarithmic phase (LP) and stationary phase (SP) promastigotes, axenic amastigotes (AA), lesion-derived amastigotes (LA), and promastigotes from the LmxMPK1 null mutant (DMPK1). Nucleic acids from 2.5 £ 107 cells were spotted per dot onto nylon membranes and probed with digoxigenin-labelled DNA probes derived from the genes indicated below the blot. MPK1–MPK9, probes derived from LmxMPK1–LmxMPK9; MBAP, probe derived from LmxMBAP. (B) Quantitative analysis of the RNA hybridisation normalised to the amount of DNA obtained from the same cells. Hybridisation intensities are shown in relative units with the highest intensity set to 100% for each probe tested. Black bar, logarithmic phase promastigotes; dark grey bar, stationary phase promastigotes; light grey bar, axenic amastigotes; white bar, lesion-derived amastigotes.

predicted using the JNet algorithm (Cuff et al., 1998; Cuff and Barton, 2000). The quality of the prediction was evaluated by comparison to the three-dimensional structure of p38 which is known at a resolution of 0.21 nm (Wang et al., 1997). Second, sequences were aligned using PILEUP, omitting the long carboxy-terminal extensions of some of the sequences. Third, alignment was modified manually to avoid the disruption of secondary structure elements by the introduction of gaps. The ATG start codons of the open reading frames are the first ATGs upstream to the DNA region encoding the conserved kinase subdomain I residues with the exception of LmxMPK7 and LmxMPK9. Here additional ATGs are located 82 and 16 nucleotides downstream to the chosen start codon respectively. However, for LmxMPK9 it is unlikely that this ATG is the naturally used start codon, because the deduced shorter

protein would lack a conserved tyrosine residue which marks the beginning of the kinase domain (indicated by an arrowhead in Fig. 4). With the exception of LmxMPK7 all start codons are the first ones following a stop codon in the respective reading frame. There are five more potential start codons in the upstream region of LmxMPK7. Determination of the splice addition site for the attachment of the capped mini-exon found at the 50 -end of each mature trypanosomatid mRNA (Laird, 1989) at a position 234 bp upstream of the first glycine codon of the GxGxxG motif in kinase subdomain I using total RNA in a RT-PCR (see Section 2) with two gene internal (50 -CCATCCACGAGATCATCGAACA-30 ; 50 -ACACCTCCCCACGCGCCAGCG-30 ) and the mini-exon primer (50 -CTAACGCTATATAAGTATCAGTTT-30 ) left us with two potential ATG start codons. Comparison to the L. major homologue of LmxMPK7 also

M. Wiese et al. / International Journal for Parasitology 33 (2003) 1577–1587

reduced the number of potential start codons to two (data not shown). It is likely that the ATG closest to the splice addition site is the naturally used start codon (Fig. 4). The nine L. mexicana MAP kinase homologues display the sequence motifs and amino acid residues characteristic for MAP kinases in their catalytic domain (Fig. 4). It starts at the position lying seven residues upstream from the first glycine in the phosphate anchor ribbon and ends in the case of protein-serine/threonine kinases 12 –18 residues downstream from the invariant arginine often displaying the four residue stretch His-Pro-(aromatic)(hydrophobic) (Hanks and Quinn, 1991). MAP kinasecharacteristic features are the subdomain I residues forming the phosphate anchor ribbon for ATP-binding with the consensus GxGxxG, the P þ 1 specificity pocket in subdomain VIII and the catalytic site residues K43, R57, R60, E61, R136, D137, K139, N142, D155 (Mg2þ ligand), R160, and T181 (numbering corresponds to LmxMPK1). The lysine corresponding to K43 of LmxMPK1 in subdomain II is conserved among all protein kinase family members (Hanks et al., 1988) and is essential for catalytic activity (Zoller and Taylor, 1979; Zoller et al., 1981; Kamps and Sefton, 1986; Buechler and Taylor, 1989; Gibbs and Zoller, 1991). It has been shown for ERK2 that mutation of this lysine residue led to a nonproductive binding of ATP suggesting that it is essential for orienting ATP for catalysis (Robinson et al., 1996). All Leishmania MAP kinase homologues contain the typical MAP kinase insert in the carboxy-terminal domain between subdomains X and XI. However, it is extended to approximately twice the size in LmxMPK7 and LmxMPK8 with insertions of 22 and 33 amino acid residues, respectively. In addition, these two proteins contain insertions in the phosphorylation lip between the DFG and the TXY motif of 42 and 62 amino acid residues, respectively (Table 2). LmxMPK7 shows a third insertion of 69 amino acid residues between the b strands b2 and b3 in the amino-terminal domain. LmxMPK6 and LmxMPK8 display long carboxy-terminal extensions of 775 and 1141 amino acid residues as compared to LmxMPK4 and LmxMPK5. LmxMPK2 and LmxMPK9 also have carboxy-terminal extensions, but of intermediate size (111 and 73 amino acid residues). There are also some additional residues (less than 50) found at the carboxy-termini of LmxMPK7 and LmxMPK3. In the region following helix ai most of the kinases carry a common docking (CD)-domain like sequence (Table 2 and Fig. 4) which has been shown to be used by MAP kinases for recognition of their activators, substrates and regulators (Tanoue et al., 2000). To further classify the nine MAP kinase homologues identified in L. mexicana, their amino acid sequences were compared to kinase signature sequences (Ku¨ltz, 1998). They all fall into the group of ERKs, in particular some of them into plant ERKs.

1583

3.4. Homology analysis of the MAP kinases Inspection of the databases led to the identification of kinases with sequence similarities to the L. mexicana MAP kinase homologues. In Trypanosoma brucei in addition to the LmxMPK1 homologue KFR1, homologues for LmxMPK2, LmxMPK4, LmxMPK5, and LmxMPK6 were found displaying amino acid identities of 58– 72% (L10997, AQ661007, Z54341, AC007863, and AF326965, accession numbers of T. brucei homologues, respectively). It is likely that the different homologues in T. brucei and the LmxMPK2 homologues in E. histolytica (accession number AT002436), P. falciparum (accession number X82646) and Dictyostelium discoideum (accession number L33043) are functionally equivalent to the L. mexicana kinases. The high percentage of amino acid identity of the L. mexicana kinases to their homologues in other Leishmania species (data not shown) is in accordance with what has been found previously for LmxMPK1 (LMPK) and its homologues in seven other Leishmania species with amino acid identities ranging from 94% to 99% (Wiese and Goercke, 2001).

4. Discussion MAP kinases are key-regulatory elements in the maintenance of the homeostasis of eukaryotic cells. Their activation or inactivation affects important processes like differentiation, proliferation and cell death. Using degenerate oligonucleotide primers in a PCR approach we identified seven new MAP kinase homologues and a cdc2related kinase homologue from L. mexicana. While subcloning and sequencing the genes, the L. major Friedlin genome sequencing project released two entire open reading frames corresponding to LmxMPK4 and LmxMPK7, and two partial sequences coding for the L. major homologues of LmxMPK3 and LmxMPK8 (AL358632, AL391562, AQ850838, and AL390935, accession numbers of L. major homologues, respectively). Moreover, a partial sequence for the L. major cdc2-related protein kinase was also included in the database (accession number AQ902628). The homologues of LmxMPK4 from Leishmania panamensis and Leishmania donovani were also sequenced and released by different researchers (accession numbers AF203877 and AF176312, respectively). Inspection of the sequences from the Leishmania genome project led to the identification of LmxMPK9 (accession number AL139796) which we missed in the PCR approach because of a serine residue in the first position of the second sequence motif which we used for primer design. We included only codons for alanine and valine in this position, which corresponds to the 30 -end of the reverse primer (BTRWYZ; B is alanine or valine; Z is arginine or serine; Table 1). Comparison of primer sequences and the sequence of LmxMPK9 showed that the only difference is the terminal nucleotide which is an adenosine in LmxMPK9 and a cytidine in the primer

1584

M. Wiese et al. / International Journal for Parasitology 33 (2003) 1577–1587

M. Wiese et al. / International Journal for Parasitology 33 (2003) 1577–1587

ATRW2. By contrast, the single clone that we obtained for LmxMPK3 allowed the non-correct base-pairing of the terminal cytidine of the primer with the adenosine present in the LmxMPK3 as part of an isoleucine codon. Despite the fact that we have not included tyrosine codons corresponding to the fourth position and leucine codons corresponding to the third position of the BTRWYZ motif we obtained LmxMPK7 and LmxCRK indicating that one mismatch preceding nine or even seven matching nucleotides at the end of a primer did not prevent the amplification of the respective open reading frame employing low-stringency PCR conditions. We determined the mRNA abundance of all nine MAP kinase homologues in logarithmic phase and stationary phase promastigotes, axenic amastigotes and lesion-derived amastigotes. Different amounts of mRNA were detected in the different life stages, indicating that the expression of these genes is at least partially regulated on the level of mRNA stability. It is reasonable to conclude that LmxMPK3 is likely to play no role in the amastigote stage as its mRNA is virtually not present. However, for all the other genes translation efficiency and protein stability could influence the amount of protein present in a given life stage independent from the amount of mRNA detected. LmxMPK6 and LmxMPK8 carry long carboxy-terminal extensions of 775 and 1141 amino acids, respectively. The extension of LmxMPK6 contains four motifs resembling SH3-binding sites with the consensus PXXP and two stretches of either four or five basic amino acids which might function as monopartite nuclear localisation signals (data not shown). LmxMPK8 contains eight putative SH3binding sites in its carboxy-terminal domain and a serinerich region from Ser(517) to Ser(562). It is likely that these two proteins are divided into an amino-terminal kinase domain and a carboxy-terminal domain which could be responsible for the localisation and/or activation of the enzyme as it is the case for the carboxy-terminal tail of ERK7 (Abe et al., 1999). On the other hand, the carboxyterminal domain could function as a scaffold for the association with interacting proteins like activators or substrates comparable to Pbs2p (Posas and Saito, 1997) or play a role as a regulatory domain in gene expression analogous to the carboxy-terminal domain of ERK5 which is a transcriptional activation domain and binding site for the transcription factor MEF2 (Kasler et al., 2000). However, no SH3-domains have been described in Leishmania to date (Parsons and Ruben, 2000).

1585

The genome of S. cerevisiae contains five MAP kinase genes plus a putative protein kinase YKL161C which clusters in a phylogenetic tree with the other MAP kinases, but contains a KXY rather than a TXY motif in its activation loop (Hunter and Plowman, 1997). In the genome of C. elegans 14 MAP kinase homologues have been identified (Plowman et al., 1999). Regarding the complexity of the organism it is likely that the nine MAP kinase homologues from L. mexicana comprise the entire set of proteins belonging to the MAP kinase family. However, more MAP kinase homologues might be identified once the genome of Leishmania is completely sequenced. Unfortunately, there is only limited information available about the function of the kinases found to display a reasonable percentage of amino acid identity to the new L. mexicana kinases. ERK2 of D. discoideum, which displays 61% of amino acid identity to LmxMPK2, has been shown to be essential for morphogenesis and cell-typespecific gene expression during multicellular development (Gaskins et al., 1996). Moreover, it is required for receptormediated activation of adenylate cyclase during aggregation (Segall et al., 1995). Adenylate cyclases have been found in T. brucei and Leishmania as molecules containing an extracellular domain with putative receptor function, a single membrane-spanning helix and a cytosolic adenylate cyclase domain (Naula and Seebeck, 2000). The genes exist as multigene families, in which members differ significantly in the extracellular portion. Adenylate cyclase activity reveals two peaks during in vitro differentiation of T. brucei from bloodstream to procyclic forms (Rolin et al., 1993). It is possible that LmxMPK2 is involved in regulation of adenylate cyclase activity affecting differentiation in Leishmania. KFR1, the T. brucei homologue of LmxMPK1, has been postulated to play a role in accelerating the proliferation of T. brucei in the mammalian blood as it is most likely involved in mediating the interferon-g-induced proliferation of the bloodstream form in the mammalian host (Hua and Wang, 1997). Likewise, LmxMPK1 has been demonstrated to be essential for the proliferation of the mammalian stage of the parasite (Wiese, 1998). Both proteins provide reasonable targets for the development of inhibitors suitable as drugs for treatment of the respective disease. TbMAPK2, a MAP kinase from T. brucei homologous to LmxMPK4, has been shown by deletion analysis to be involved in the differentiation of bloodstream form to procyclic form trypanosomes which displayed a block in cell cycle progression in all phases of the cell cycle

Fig. 4. Multiple sequence alignment of nine mitogen-activated protein kinase homologues of L. mexicana with p38. LmxMPK1– 9, L. mexicana mitogenactivated protein kinase. Black bars I –XI indicate mitogen-activated protein kinase typical subdomains. Secondary structure element amino acid residues are highlighted; b-sheets, grey; a-helices, black. Arrowheads mark the borders of the kinase core structure; open circles, phosphate anchor ribbon (Gly21–Gly26); triangle, conserved lysine which leads to loss of kinase activity after mutation; filled circles, conserved amino acid residues in Ser/Thr-protein kinases; crossed circles, P þ 1 specificity pocket (Ala180–Arg185); arrows, potential regulatory phosphorylation sites; underlined amino acid residues, potential common docking-domain. Dashes indicate gaps introduced to optimise the alignment; dots represent identical amino acid residues as in the top line; asterisks mark the carboxy-terminus of the proteins. Lengths of amino acid insertions and carboxy-terminal extensions are indicated in parentheses at the respective positions. Numbering corresponds to LmxMPK1.

1586

M. Wiese et al. / International Journal for Parasitology 33 (2003) 1577–1587

(Mu¨ller et al., 2002). In the future, the new MAP kinase homologues will be evaluated as potential drug targets in deletion experiments. Moreover, the identification of kinase-specific amino acids and sequence motifs enables us to study the function of these molecules using mutated versions of the kinases. It is likely that all MAP kinase homologues of Leishmania have a counterpart in Trypanosoma since already five homologues are present in the T. brucei genome database. Therefore, each potential drug target identified in Leishmania points out the corresponding molecule in Trypanosoma and vice versa increasing the efficiency of target identification and the development of new remedies against the diseases caused by these organisms.

Acknowledgements We thank Tanja Herrmann for excellent help in subcloning and sequencing, and Dr Peter Overath and Daniela Kuhn for critically reading the manuscript.

References Abe, M.K., Kuo, W.L., Hershenson, M.B., Rosner, M.R., 1999. Extracellular signal-regulated kinase 7 (ERK7), a novel ERK with a C-terminal domain that regulates its activity, its cellular localisation, and cell growth. Mol. Cell. Biol. 19, 1301–1312. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Bellon, S., Fitzgibbon, M.J., Fox, T., Hsiao, H.M., Wilson, K.P., 1999. The structure of phosphorylated p38gamma is monomeric and reveals a conserved activation-loop conformation. Struct. Fold. Des. 7, 1057–1065. Buechler, J.A., Taylor, S.S., 1989. Dicyclohexylcarbodiimide cross-links two conserved residues, Asp-184 and Lys-72, at the active site of the catalytic subunit of cAMP-dependent protein kinase. Biochemistry 28, 2065–2070. Canagarajah, B.J., Khokhlatchev, A., Cobb, M.H., Goldsmith, E.J., 1997. Activation mechanism of the MAP kinase ERK2 by dual phosphorylation. Cell 90, 859–869. Cobb, M.H., Goldsmith, E.J., 2000. Dimerization in MAP-kinase signaling. Trends Biochem. Sci. 25, 7–9. Cuff, J.A., Barton, G.J., 2000. Application of multiple sequence alignment profiles to improve protein secondary structure prediction. Proteins 40, 502– 511. Cuff, J.A., Clamp, M.E., Siddiqui, A.S., Finlay, M., Barton, G.J., 1998. JPred: a consensus secondary structure prediction server. Bioinformatics 14, 892–893. Devereux, J., Haeberli, P., Smithies, O., 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387– 395. Garrington, T.P., Johnson, G.L., 1999. Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr. Opin. Cell Biol. 11, 211– 218. Gaskins, C., Clark, A.M., Aubry, L., Segall, J.E., Firtel, R.A., 1996. The Dictyostelium MAP kinase ERK2 regulates multiple, independent developmental pathways. Genes Dev. 10, 118–128.

Gibbs, C.S., Zoller, M.J., 1991. Rational scanning mutagenesis of a protein kinase identifies functional regions involved in catalysis and substrate interactions. J. Biol. Chem. 266, 8923–8931. Hanks, S.K., Hunter, T., 1995. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 9, 576– 596. Hanks, S.K., Quinn, A.M., 1991. Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. Methods Enzymol. 200, 38– 62. Hanks, S.K., Quinn, A.M., Hunter, T., 1988. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241, 42–52. Hua, S.B., Wang, C.C., 1997. Interferon-gamma activation of a mitogenactivated protein kinase, KFR1, in the bloodstream form of Trypanosoma brucei. J. Biol. Chem. 272, 10797–10803. Hunter, T., Plowman, G.D., 1997. The protein kinases of budding yeast: six score and more. Trends Biochem. Sci. 22, 18–22. Kamps, M.P., Sefton, B.M., 1986. Neither arginine nor histidine can carry out the function of lysine-295 in the ATP-binding site of p60src. Mol. Cell. Biol. 6, 751–757. Kasler, H.G., Victoria, J., Duramad, O., Winoto, A., 2000. ERK5 is a novel type of mitogen-activated protein kinase containing a transcriptional activation domain. Mol. Cell. Biol. 20, 8382–8389. Ku¨ltz, D., 1998. Phylogenetic and functional classification of mitogen- and stress-activated protein kinases. J. Mol. Evol. 46, 571–588. Laird, A.D., Vajkoczy, P., Shawver, L.K., Thurnher, A., Liang, C., Mohammadi, M., Schlessinger, J., Ullrich, A., Hubbard, S.R., Blake, R.A., Fong, T.A.T., Strawn, L.M., Sun, L., Tang, C., Hawtin, R., Tang, F., Shenoy, N., Hirth, K.P., McMahon, G., Cherrington, J.M., 2000. SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors. Cancer Res. 60, 4152–4160. Laird, P.W., 1989. Trans splicing in trypanosomes – archaism or adaptation? Trends Genet. 5, 204–208. Lee, J.C., Laydon, J.T., McDonnell, P.C., Gallagher, T.F., Kumar, S., Green, D., McNulty, D., Blumenthal, M.J., Heys, J.R., Landvatter, S.W., 1994. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372, 739 –746. Medina-Acosta, E., Cross, G.A., 1993. Rapid isolation of DNA from trypanosomatid protozoa using a simple ‘mini-prep’ procedure. Mol. Biochem. Parasitol. 59, 327 –329. Menz, B., Winter, G., Ilg, T., Lottspeich, F., Overath, P., 1991. Purification and characterization of a membrane-bound acid phosphatase of Leishmania mexicana. Mol. Biochem. Parasitol. 47, 101–108. Morgan, D.O., 1995. Principles of CDK regulation. Nature 374, 131 –134. Mu¨ller, I.B., Domenicali-Pfister, D., Roditi, I., Vassella, E., 2002. Stagespecific requirement of a mitogen-activated protein kinase by Trypanosoma brucei. Mol. Biol. Cell 13, 3787–3799. Naula, C., Seebeck, T., 2000. Cyclic AMP signaling in trypanosomatids. Parasitol. Today 16, 35 –38. Parsons, M., Ruben, L., 2000. Pathways involved in environmental sensing in trypanosomatids. Parasitol. Today 16, 56–62. Pearson, G., Robinson, F., Gibson, T.B., Xu, B.E., Karandikar, M., Berman, K., Cobb, M.H., 2001. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr. Rev. 22, 153 –183. Plowman, G.D., Sudarsanam, S., Bingham, J., Whyte, D., Hunter, T., 1999. The protein kinases of Caenorhabditis elegans: a model for signal transduction in multicellular organisms. Proc. Natl. Acad. Sci. USA 96, 13603–13610. Posas, F., Saito, H., 1997. Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: scaffold role of Pbs2p MAPKK. Science 276, 1702–1705. Robinson, M.J., Harkins, P.C., Zhang, J., Baer, R., Haycock, J.W., Cobb, M.H., Goldsmith, E.J., 1996. Mutation of position 52 in ERK2 creates a nonproductive binding mode for adenosine 50 -triphosphate. Biochemistry 35, 5641–5646.

M. Wiese et al. / International Journal for Parasitology 33 (2003) 1577–1587 Rolin, S., Paindavoine, P., Hanocq-Quertier, J., Hanocq, F., Claes, Y., Le Ray, D., Overath, P., Pays, E., 1993. Transient adenylate cyclase activation accompanies differentiation of Trypanosoma brucei from bloodstream to procyclic forms. Mol. Biochem. Parasitol. 61, 115–125. Schaeffer, H.J., Weber, M.J., 1999. Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol. Cell. Biol. 19, 2435–2444. Segall, J.E., Kuspa, A., Shaulsky, G., Ecke, M., Maeda, M., Gaskins, C., Firtel, R.A., Loomis, W.F., 1995. A MAP kinase necessary for receptormediated activation of adenylyl cyclase in Dictyostelium. J. Cell Biol. 128, 405 –413. Tanoue, T., Adachi, M., Moriguchi, T., Nishida, E., 2000. A conserved docking motif in MAP kinases common to substrates, activators and regulators. Nat. Cell Biol. 2, 110–116. Tong, L., Pav, S., White, D.M., Rogers, S., Crane, K.M., Cywin, C.L., Brown, M.L., Pargellis, C.A., 1997. A highly specific inhibitor of human p38 MAP kinase binds in the ATP pocket. Nat. Struct. Biol. 4, 311–316. Wang, Z., Harkins, P.C., Ulevitch, R.J., Han, J., Cobb, M.H., Goldsmith, E.J., 1997. The structure of mitogen-activated protein kinase p38 at 2.1A resolution. Proc. Natl. Acad. Sci. USA 94, 2327– 2332. Wiese, M., 1998. A mitogen-activated protein (MAP) kinase homologue of Leishmania mexicana is essential for parasite survival in the infected host. EMBO J. 17, 2619– 2628. Wiese, M., Goercke, I., 2001. Homologues of LMPK, a mitogen-activated protein kinase from Leishmania mexicana, in different Leishmania species. Med. Microbiol. Immunol. 190, 19–22. Wiese, M., Ilg, T., Lottspeich, F., Overath, P., 1995. Ser/Thr-rich repetitive motifs as targets for phosphoglycan modifications in Leishmania mexicana secreted acid phosphatase. EMBO J. 14, 1067–1074.

1587

Wiese, M., Berger, O., Stierhof, Y.-D., Wolfram, M., Fuchs, M., Overath, P., 1996. Gene cloning and cellular localisation of a membrane-bound acid phosphatase of Leishmania mexicana. Mol. Biochem. Parasitol. 82, 153– 165. Wilson, K.P., Fitzgibbon, M.J., Caron, P.R., Griffith, J.P., Chen, W., McCaffrey, P.G., Chambers, S.P., Su, M.S., 1996. Crystal structure of p38 mitogen-activated protein kinase. J. Biol. Chem. 271, 27696–27700. Wilson, K.P., McCaffrey, P.G., Hsiao, K., Pazhanisamy, S., Galullo, V., Bemis, G.W., Fitzgibbon, M.J., Caron, P.R., Murcko, M.A., Su, M.S., 1997. The structural basis for the specificity of pyridinylimidazole inhibitors of p38 MAP kinase. Chem. Biol. 4, 423 –431. Xie, X., Gu, Y., Fox, T., Coll, J.T., Fleming, M.A., Markland, W., Caron, P.R., Wilson, K.P., Su, M.S., 1998. Crystal structure of JNK3: a kinase implicated in neuronal apoptosis. Structure 6, 983 –991. Zhang, F., Strand, A., Robbins, D., Cobb, M.H., Goldsmith, E.J., 1994. Atomic structure of the MAP kinase ERK2 at 2.3 A resolution. Nature 367, 704–711. Zhou, G.C., Bao, Z.Q., Dixon, J.E., 1995. Components of a new human protein-kinase signal-transduction pathway. J. Biol. Chem. 270, 12665–12669. Zoller, M.J., Taylor, S.S., 1979. Affinity labeling of the nucleotide binding site of the catalytic subunit of cAMP-dependent protein kinase using pfluorosulfonyl-{14C}benzoyl 50 -adenosine. Identification of a modified lysine residue. J. Biol. Chem. 254, 8363–8368. Zoller, M.J., Nelson, N.C., Taylor, S.S., 1981. Affinity labeling of cAMP-dependent protein kinase with p-fluorosulfonylbenzoyl adenosine. Covalent modification of lysine 71. J. Biol. Chem. 256, 10837–10842.