Molecular cloning and characterisation of SmSLK, a novel Ste20-like kinase in Schistosoma mansoni

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International Journal for Parasitology 37 (2007) 1539–1550

Molecular cloning and characterisation of SmSLK, a novelSte20-like kinase in Schistosoma mansoni q

Yutao Yan a,1, David Tulasne b, Edith Browaeys c, Katia Cailliau c, Naji Khayath a,Raymond J. Pierce a, Jacques Trolet a, Veronique Fafeur b, Amena Ben Younes d,

Colette Dissous a,*

a Inserm, U 547, Lille, France; Universite Lille 2, Lille, France; Institut Pasteur de Lille, IFR142, 1 rue du Professeur A. Calmette, 59019-Lille, Franceb CNRS UMR 8161, Institut de Biologie de Lille/Institut Pasteur de Lille, 1 Rue Pr Calmette, BP447, 59021-Lille Cedex, France

c Universite des Sciences et Technologies de Lille, Laboratoire de Regulation des Signaux de Division EA 4020, SN3, 59655 Villeneuve d’Ascq Cedex, Franced IFR 142 Inserm, Institut Pasteur de Lille, France

Received 26 April 2007; received in revised form 5 June 2007; accepted 8 June 2007

Abstract

Serine/threonine kinases of the Ste20 group play important roles in various cellular functions such as growth, apoptosis and morpho-genesis. This family includes p21-Activated Kinases (PAKs) and Germinal Center Kinases (GCKs) families which contain their kinasedomain in the C-terminal and N-terminal position, respectively. Here, we report the characterisation of a novel Ste20-like kinase (SLK)in the helminth parasite Schistosoma mansoni (SmSLK). SmSLK belongs to the GCK subfamily and contains a conserved N-terminalSte20-like catalytic domain and C-terminal coiled-coil structures homologous to mammalian Lymphocyte Oriented Kinase (LOK) andSLK kinases and described as regulatory domains in these proteins. Gene assembly was performed using S. mansoni sequences availablefrom genomic databases and indicated that SmSLK is composed of 18 exons and present in one copy in the S. mansoni genome. RT-PCRexperiments demonstrated an alternative splicing of SmSLK in the exon 9 encoding the hinge region between kinase and coiled-coildomains of SmSLK and showed the expression of both transcript isoforms (SmSLK and SmSLK-S in which exon 9 is deleted) in allthe S. mansoni parasite stages. Most of the Ste20-related proteins are active kinases known to regulate mitogen-activated protein kinase(MAPK) cascades. We demonstrated the kinase activity of SmSLK and SmSLK-S and their capacity to activate the MAPK/Jun N-ter-minal kinase (JNK) pathway in human embryonic kidney (HEK) cells as well as in Xenopus oocytes. Immunofluorescence studies indi-cated that SmSLK proteins were abundant in the tegument of adult schistosomes. Therefore, these results indicate that SmSLK is a newmember of the GCK protein family that could participate in the regulation of MAPK cascade activation during host–parasiteinteractions.� 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Ste20 kinase; Schistosoma mansoni; Mitogen-activated protein kinase; Signalling pathway

0020-7519/$30.00 � 2007 Australian Society for Parasitology Inc. Published b

doi:10.1016/j.ijpara.2007.06.001

q Note: Nucleotide sequence data reported in this paper are available inthe GenBank� database, Accession No. AY149571.

* Corresponding author. Tel.: +33 3 20 87 73 50; fax: +33 3 20 87 78 88.E-mail address: [email protected] (C. Dissous).

1 Present address: Division Digestive Diseases, Department of Medicine,Emory University, Whitehead Building, 615 Michael Street, Atlanta, GA30322, USA.

1. Introduction

Schistosomiasis is a major helminth infection which isan important public concern in many developing countries.It is the second major worldwide parasitic disease aftermalaria, affecting 200 million individuals and responsiblefor 280,000 deaths annually (van der Werf et al., 2003).Schistosomes are digenean parasites genetically pro-grammed to develop sequentially within mollusc intermedi-ate and vertebrate definitive hosts. They have a complex

y Elsevier Ltd. All rights reserved.

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1540 Y. Yan et al. / International Journal for Parasitology 37 (2007) 1539–1550

life cycle with different morphological stages and theirdevelopment requires permanent processes of communica-tion with their host environment.

Intracellular signalling cascades are the main routes ofcommunication between the plasma membrane and regula-tory targets of intracellular compartments. Recent studiesaiming at a better understanding of development and dif-ferentiation processes in Schistosoma mansoni have led tothe identification of several conserved receptor and cyto-solic protein kinases in the parasite. Receptor tyrosinekinases homologous to EGF (Shoemaker et al., 1992; Vico-gne et al., 2004) and insulin (Khayath et al., 2007) receptorsand members of the TGF-b receptor family (Davies et al.,1998; Forrester et al., 2004; Osman et al., 2006) have beenfunctionally characterised in S. mansoni, as well as diversecellular tyrosine kinases of the Src (Kapp et al., 2004) andSyk (Knobloch et al., 2002) families or homologous to Fynproteins (Kapp et al., 2001).

Extracellular stimuli acting on growth factor or G-pro-tein coupled receptors transduce most of their effectsthrough mitogen-activated protein kinase (MAPK) signal-ling cascades to the nucleus, initiating different cellular pro-cesses such as proliferation, differentiation, developmentand stress response apoptosis. The signalling is mediatedby sequential phosphorylation of a multiple-kinase moduleinvolving at least three levels of protein kinases. In multi-cellular organisms, distinct MAPK pathways exist amongwhich the ERK, Jun N-terminal kinase (JNK) and p38 cas-cades are the best known. MAPKs have been shown to beexpressed in S. mansoni (Schussler et al., 1997) and a largenumber of MAPK pathway components were recentlyfound in the Schistosoma japonicum genome using bioinfor-matics approaches (Wang et al., 2006).

In Saccharomyces cerevisiae, the Ste20 (Sterile-20) ser-ine/threonine (Ser/Thr) protein kinase was originally foundto be a key kinase in the mating pathway, initiated by thebinding of a peptide pheromone to its receptor and involv-ing the activation of the MAPK pathway composed ofSte11p (MAP3K), Ste7p (MAP2K) and Fus3p/Kss1p(MAPKs). By direct phosphorylation of Ste11p, Ste20pacts as a mitogen-activated protein kinase kinase kinasekinase (MAP4K) in the yeast pathway (Wu et al., 1995;Drogen et al., 2000). A large group of Ste20-related kinaseswas further identified in mammals and other organismsand most of these kinases were also demonstrated to regu-late MAPK cascades. Moreover, kinases of Ste20 familyplay important roles in various cellular functions such asgrowth, apoptosis and morphogenesis

Ste20-related kinases are characterised by a conservedSer/Thr kinase domain and a non-catalytic region withconsiderable structural and sequence diversity which allowsthem to interact with various cytoskeleton regulatory pro-teins (see Dan et al., 2001). This kinase group comprisestwo distinct families, p21-activated kinases (PAKs) andgerminal center kinases (GCKs), which largely differ intheir structural organisation and specificity of function.PAKs contain their catalytic domain within the C-terminus

and a Cdc42/Rac interactive binding (CRIB) domainwithin the N-terminus. Upon binding to guanosine triphos-phatases Cdc42 and/or Rac, PAKs undergo a conforma-tional change which enables autophosphorylation andsubsequent kinase activation. They are mostly active onthe cytoskeleton and are involved in cell motility and mor-phology. GCKs are constituted by an N-terminal kinasedomain and a variable C-terminal regulatory domainwhich has been used to classify GCKs in different subclass-es. Analysis of phylogenetic relationships among mamma-lian Ste20-related kinases distinguished at least eight GCKsubfamilies, characterised by their capacity to interact withspecific partners of various signalling pathways (Dan et al.,2001).

In this paper, we believe we describe the first character-isation of an Ste20-related protein in the parasite S. man-

soni. This protein named SmSLK (for S. mansoni Ste20-Like Kinase) has a conserved GCK structure homologousto the previously characterised mammalian LymphocyteOriented Kinase (LOK) and SLK proteins. Two isoformsof SmSLK exist and are expressed at all the life-cycle stagesof the parasite. Their functional kinase activity was demon-strated as well as their capacity to activate the JNK/MAPK pathway in vertebrate cells. These results indicatethat SmSLK is a new member of the GCK protein family.

2. Materials and methods

2.1. Parasite material

A Puerto-Rican strain of S. mansoni was maintained bypassage through albino Biomphalaria glabrata snails andMesocricetus auratus hamsters. Miracidia and cercariaewere prepared as previously described (Dissous andCapron, 1981). Miracidia were transformed into sporocystsin vitro by incubation in minimum salt medium at 28 �Cfor 18 h (Schallig et al., 1990). Adult schistosomes were col-lected by portal perfusion from infected hamsters. TotalRNA was isolated by the method of Chirgwin et al.(1979) and purified by centrifugation through a caesiumchloride gradient.

2.2. Molecular cloning of SmSLK

First-strand cDNA was synthesised from adult wormtotal RNA using the Thermoscript� RT-PCR System(Invitrogen) and the oligo(dT)20 primer. Three degenerateoligonucleotide primers (5 0GGN III GGN WSN TTYGGN ATG GT NTA 3 0; 5 0 CAN ARR CAR TTN CKNGCN GCN ARR TCN CKR TGD AT 3 0 and 5 0 DATYTC CCA NAR NAC N AC NCC RTA NWS CCA 3 0)encoding, respectively, the conserved motifsGXGSFGMVY, IHRDLAARCLV and WSYGVVLWEIclassically contained in kinase catalytic domains (Hanksand Hunter, 1995), were used as primers in RT-PCR toamplify schistosome cDNA. PCR products were cloned intothe pCR2.1-TOPO vector (Invitrogen), sequenced using the

Y. Yan et al. / International Journal for Parasitology 37 (2007) 1539–1550 1541

Big-Dye Terminator cycle sequencing kit (Applied Biosys-tems) and analysed on an ABI Prism 377 DNA sequencer(Applied Biosystems). The full-length cDNA sequence(GenBank No. AY149571) of SmSLK was obtained byamplification of 5 0 rapid amplification of cDNA ends(RACE) and 3 0 RACE cDNAs synthesised using theSMART RACE cDNA amplification kit (Clontech, CA)according to the manufacturer’s instructions. In these exper-iments, a shorter isoform of the protein (SmSLK-S) wasidentified. Sequence analysis of SmSLK and SmSLK-Swas performed using programs included in the Lasergenepackage (DNASTAR Inc., Madison, WI, USA).

2.3. SmSLK plasmid constructs

SmSLK forward, 5 0GCC CGA AAT GCT AAA ATCGTT CTT TAA G 3 0 and SmSLK reverse, 5 0 TCA CATTGA ATC AGT GGA ACC TGT TTT 3 0 primers comple-mentary to the N and C-terminal sequences of the SmSLKcDNAs were completed with 5 0 NotI restriction sites andused in RT-PCR experiments to amplify SmSLK andSmSLK-S full length cDNAs. PCR were carried out withplatinum Taq DNA polymerase high fidelity (Invitrogen).Final products were purified, cloned into the pCR4-TOPOvector (Invitrogen), then inserted in frame into the NotI siteof the V5/His-tagged vector pcDNA3.1 expression vector(Invitrogen). SmSLK and SmSLK-S-pcDNA V5 constructswere sequenced to control the absence of nucleotide muta-tion. Dead kinase constructs (SmSLKDK and SmSLK-SDK-pcDNA V5) were further obtained by site-directedmutagenesis of the D190FG192 active motif into a DNA inac-tive motif using the QuickChange� site-directed mutagene-sis kit (stratagene) and the 5 0 GAA GTT AAA CTT GCTGAT AAT GCA GTT TCT GCC AAG TTA 3 0 mutatedsequence and its reverse complement as primers. MouseSLK myc-tagged construct (mSLK-pcDNA myc) was a giftfrom L.A. Sabourin (McMaster University, Ont., Canada).

2.4. Northern blot analysis

Total RNA (20 lg) of S. mansoni adult worms was sep-arated on a formaldehyde agarose gel, blotted ontoHybond-N+ nylon charged membrane (Amersham), fixedby alkali treatment (in 50 mM NaOH) and prehybridisedat 42 �C for 3 h. A cDNA fragment (comprising nucleo-tides 863–1832 of the SmSLK sequence and common toSmSLK-S) was labelled with [32P]a-dCTP (MegaprimeDNA labelling system kit, Amersham) and used forhybridisation to detect both SmSLK and SmSLK-S tran-scripts. Another cDNA fragment complementary to thealternative exon only present in SmSLK (comprising nucle-otides 1285–1515 of SmSLK) was labelled under the sameconditions and used as a probe to detect SmSLK tran-scripts selectively. Hybridisation was performed at 42 �Cfor 18 h in 5· sodium chloride–sodium citrate (SSC), 5·Denhardt’s reagent, 0.5% SDS, 0.1 mg ml�1 denatured her-ring sperm DNA and 50% formamide. The membrane was

washed twice in 2· SSC, 0.1% SDS for 10 min at room tem-perature, once in 0.1· SSC, 0.1% SDS for 10 min at 42 �Cand was then autoradiographed.

2.5. Quantitative RT-PCR

Total RNA extracted from miracidia, sporocysts, cerca-riae and adult male and female worms was reverse tran-scribed using the Thermoscript� RT-PCR System(Invitrogen). cDNAs were used as templates for PCRamplification using the SYBR� Green PCR Master Mixand the ABI PRISM 7000 sequence detection system(Applied Biosystems). Two sets of primers were designedby the Primer Express Program (Applied Biosystems)which enabled us to discriminate between the amplificationof SmSLK and SmSLK-S. Two primers hybridizing toboth SmSLK and SmSLK-S isoforms (positions 1728–1753; 1829–1803 in the SmSLK sequence) were used toamplify total transcripts of SmSLK and SmSLK-S in trip-licate assays. Another set of two primers specific for thealternative exon only present in SmSLK (positions1447–1467; 1547–1526 in the SmSLK sequence) was usedto specifically amplify SmSLK transcripts under similarconditions. Primers specific for S. mansoni tubulin(GenBank Accession No. M80214, positions 851–873;925–904) were used as internal controls. For graphical rep-resentation of total SmSLK transcript quantitative PCR(qPCR) data, the raw cycle threshold (DCt values) obtainedfor female worms, miracidia, sporocysts and cercariae werededucted from the DCt value obtained for male worm tran-script levels using the delta–delta Ct (DDCt) method (Livakand Schmittgen, 2001), with tubulin gene levels serving asthe internal standard. Efficiency rates of the PCR reactionswere uniformly high and comparable (�2), permittinganalysis by this method. To analyse the respective amountsof SmSLK and SmSLK-S in a given parasite stage, DCt

values obtained when using SmSLK exon-specific primerswere deducted from those obtained using primers commonto both SmSLK isoforms, then analysed using the samemethod.

2.6. Genomic analysis

In order to determine the SmSlk gene structure, genomicsequence data comprising individual shotgun sequencingreads and contigs available from the Wellcome Trust San-ger Institute website were subjected to BLASTN analysiswith the SmSLK cDNA sequence (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/s_mansoni). Align-ments and exon–intron junctions were verified usingprograms included in the Lasergene package.

2.7. Expression of SmSLK proteins in Xenopus oocytes

Capped messenger RNA (cRNA) encoding the differentSmSLK proteins was synthesised in vitro using the T7mMessage mMachine Kit (Ambion, USA). SmSLK,

http://www.sanger.ac.uk/cgi-bin/blast/submitblast/s_mansoni

http://www.sanger.ac.uk/cgi-bin/blast/submitblast/s_mansoni


SmSLK-S, SmSLKDK and SmSLK-SDK-pcDNA V5 plas-mids were linearised by the enzyme PmeI. cRNAs tran-scribed from 1 lg of each linearised plasmid wereprecipitated by 2.5 M LiCl, washed in 70% ethanol, resus-pended in 20 ll diethyl pyrocarbonate (DEPC)-treatedwater, then quantified by spectrophotometry. Finally,1 lg of cRNA was analysed on a denaturating agarosegel. Gel staining with 10 lg ml�1 ethidium bromide con-firmed the size of cRNA and verified the absence of abor-tive transcripts. cRNA preparations were microinjectedinto Xenopus oocytes according to the protocol previouslydescribed (Vicogne et al., 2004). After anaesthesia with MS222 (1 g l�1, Sandoz), ovarian fragments of Xenopus laevis

were surgically removed and placed in ND96 medium(96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2,5 mM N-2-hydroxyethylpiperazine-N 0-2-ethanesulfonicacid (HEPES) adjusted to pH 7.4 with NaOH), supple-mented with streptomycin/penicillin (50 lg ml�1 each,Eurobio), sodium pyruvate (225 lg ml�1, Sigma) and soy-bean trypsin inhibitor (30 lg ml�1, Sigma). Stage VIoocytes were harvested following 1 h treatment with1 mg ml�1 collagenase A (Roche Applied Science). Com-plete defolliculation of oocytes was achieved by manualdissection. Oocytes were kept at 19 �C in ND96 mediumbefore microinjection in the equatorial region. In eachassay, 20–30 oocytes removed from at least two differentanimals were used. Sixty nanolitres (equivalent to 60 ng)of wild or mutant SmSLK and SmSLK-S cRNA wereinjected into each oocyte. Oocyte homogenates were ana-lysed by Western blotting as described previously (Browa-eys-Poly et al., 2000). Blots were incubated either with anti-V5 antibodies (1/1000, Invitrogen), anti-c JNK antibodies(1/1000, Sigma) or anti-phosphoactive JNK antibodies(1/800, Promega). Horseradish-peroxidase (HRP)-labelledsecondary antibodies were detected using the enhancedchemiluminescence Western blotting detection system(Amersham).

2.8. Cell culture and transfection

Human embryonic kidney (HEK293T) cells were main-tained in Dulbecco’s modified eagle medium (DMEM) sup-plemented with 10% FCS in a humidified atmosphere of5% CO2 in air at 37 �C. For transfection experiments, cellswere seeded in 6-well plates (4· 105 cells per well) 1 dayprior to transfection. At 75% of confluence, cells werewashed and placed in OptiMEM just before transfectionby plasmid DNA using Exgen500 (Euromedex) as a trans-fection reagent. For each well, 6 ll of Exgen500 were mixedwith 1 lg of plasmid DNA (SmSLK, SmSLK-S,SmSLKDK and SmSLK-SDK-pcDNA V5, mSLK- pcDNAmyc or empty pcDNA3 as control) in a total volume of100 ll OptiMEM, incubated at room temperature for20 min and then added to the cells. Following an incuba-tion of 4 h, cells were washed and incubated in DMEM/FCS for an additional period of 24 h. In experimentsdevoted to MAPK activation assays, HEK293T cells were

co-transfected either with SmSLK-pcDNA V5, SmSLK-S-pcDNA V5 or mSLK-pcDNA myc plasmids together with1 lg of pcDNA vector encoding FLAG-tagged JNK-1kinase (Paumelle et al., 2000) under the same conditions.

2.9. Immunoprecipitations and in vitro kinase assays

Transfected cells were washed twice in PBS pH 7.4, thenlysed in 300 ll lysis buffer (50 mM Tris pH 7.8, 150 mMNaCl, 1% Nonidet P40) and placed on ice for 30 min.Lysates were immunoprecipitated by anti-V5 or anti-mycantibodies under the conditions previously described(Tulasne et al., 1999). Immune complexes bound to proteinA-Sepharose beads were washed three times with lysis buf-fer, then twice with kinase buffer consisting of 50 mMHEPES, pH 7.5, 12.5 mM MgCl2, 150 mM NaCl, 1 mMdithiothreitol (DTT), 1 mM Na3VO4 and 1 mM phenyl-methylsulfonyl fluoride (PMSF). Kinase activity ofexpressed proteins was analysed using myelin basic protein(MBP) as an exogenous phosphorylation substrate. Briefly,3 lg of MBP were added to immune complexes and incu-bated in a total volume of 30 ll kinase buffer in thepresence of 10 lCi [c-32P]ATP (diluted with cold ATP to givea final concentration of 200 lM) at 30 �C for 30 min. Reac-tions were stopped by the addition of 8 ll of 5· SDS–PAGEsample buffer and heated at 100 �C for 3 min. Eluates wererecovered by centrifugation and analysed on a4–12% gradient SDS–polyacrylamide gel. Gels were fixed,dried, and phosphorylated substrates were detected by auto-radiography. A positive control of substrate phosphoryla-tion was obtained by incubation of MBP with purifiedrecombinant Abl protein tyrosine kinase (Calbiochem) asper the manufacturer’s instructions.

The JNK activity assays were performed under similarkinase reaction conditions. In these assays, lysates fromcells co-transfected with SLK-encoding plasmids andFLAG-tagged JNK-1 kinase plasmid were immunoprecip-itated by an anti-FLAG antibody (M2, Sigma) and kinasereactions were performed on anti-FLAG immunoprecipi-tates using 3 lg of purified GST-c-jun protein (Paumelleet al., 2000) as a specific substrate. The detection of radio-active c-jun proteins was performed by autoradiography asdescribed above.

2.10. Immunolocalisation of SmSLK proteins

Antibodies were produced against the non-conservedprotein domain of SmSLK (amino acids 289–612 ofSmSLK) subcloned in the PQE 30 expression vector andexpressed in Escherichia coli. Antisera were produced inmice after immunisation against the recombinant proteinwith FCA under the conditions previously described(Khayath et al., 2007).

Male adult worms were fixed for 1 h at room tempera-ture in 2.5% paraformaldehyde containing 0.1% glutaralde-hyde, and then washed in 10 mM phosphate buffer (PB) pH7.4 containing 0.1 M glycine. Worms were immobilised in


2% agarose, post-fixed in 0.5% uranyl acetate in Michaelisbuffer, dehydrated in ethanol together with temperaturedecrease and embedded in LW HM20 at progressive lowtemperature ‘‘PLT’’ (Gounon, 1999). Immunolocalisationwas performed on 300 nm-thick sections according to themodified procedure of Spehner et al. (2002). Briefly, sam-ples loaded on 3-aminopropyltriethoxy-silane coated slideswere hydrated in PB, and then in PBS containing 0.1 Mglycine. Endogenous fluorescence was inhibited with50 mM NH4Cl and non-specific sites were blocked withPBS containing 3% milk and 0.05% tween 20. Samples werethen incubated with anti-SmSLK mouse serum (1/50) orwith normal mouse serum as a negative control. Boundantibodies were revealed by using (Fab 0)2 goat anti-mouseantibodies conjugated to Alexa 488 fluorochrome at a 1/500 dilution (Jackson Immunoresearch Laboratories, WestGrove, PA). Slides were mounted with Fluoromount G(Southern Biotechnology Assoc. Birmingham, Al) andexamined by using a Leica microscope fitted with fluores-cence appropriate filters and a Cool SNAP colour CCDcamera (Princeton instruments-Gatan, Roper Scientific,Germany).

3. Results

3.1. Molecular cloning and characterisation of SmSLK

Degenerate RT-PCR, used for the discovery of newmembers of the kinase family in S. mansoni, led to the iso-lation of a partial cDNA encoding a novel protein kinase.To obtain the full-length sequence, several 5 0-RACE and3 0-RACE experiments were performed successively, andfinally a PCR product of 3710 bp was amplified which con-tained a 3168 bp open reading frame encoding a protein of1056 residues with a predicted molecular mass of 122 kDaand a theoretical pI of 6.51. Blast searches revealedsequence similarity of this protein with members of theSLK family.

The protein, termed SmSLK, contains an N-terminalkinase domain and a large C-terminal domain, and there-fore potentially belongs to the GCK subfamily of Ste20-like proteins (Fig. 1). The catalytic domain of SmSLKextending up to residue 295, was shown to contain the 11conserved subdomains of Ser/Thr kinases (Hanks andHunter, 1995) (Fig. 1a) and its sequence showed significantidentity (about 50%) to mouse SLK (Sabourin and Rudn-icki, 1999) and LOK proteins (Kuramochi et al., 1997,1999) as well as to the Xenopus polo-like kinase kinase 1(xPlkk1) (Qian et al., 1998), three other members of theGCK subfamily. Subdomain I contains the consensussequence G41-X-G-X-X-G46 that is essential for ATP bind-ing, as well as the invariant V48 necessary for the correctpositioning of conserved glycine residues and their interac-tion with the ATP molecule. The conserved lysine residuewithin the ATP binding site of subdomain II was foundat position 63. In the kinase subdomain VIII, the Ste20 sig-

nature motif IGTPYWMAPEV characteristic of SLK fam-ily members was present (Dan et al., 2001).

Sequence alignment of the non-catalytic domains ofSmSLK with related proteins also revealed a high level ofsimilarity to the C-termini of mouse LOK and xPlkk1 com-posed of coiled-coil structures similar to that found in theamino acid sequence of rat AT1-46, a protein preferentiallyexpressed in the olfactory-limbic system of adult rat brainand composed of a-helices predicted to be arranged incoiled-coils (Schaar et al., 1996) (Fig. 1b). Using ‘‘COILS’’(Swiss EMBnet node server; Lupas et al., 1991), a programthat predicts coiled-coil regions, we could demonstrate thatthe SmSLK sequence from residues 560 to 984 also pre-sented a high probability to adopt a coiled-coil conforma-tion. Two coiled-coil domains spanning from positions608–708 and 888–959 in SmSLK were identified using thisprogram (Fig. 1c). All these data confirmed that SmSLKwas a new member of the GCK family, homologous toLOK proteins that belong to the GCK-V subfamily accord-ing to the phylogenetic classification of Dan et al. (2001).

3.2. Existence of two different SmSLK transcripts

Further amplification of adult worm cDNA led us toisolate a spliced variant of SmSLK with a 231 bp nucleo-tide in-frame deletion (nucleotides 1285–1515, residue412–488) in the region located between the kinase domainand the coiled-coil structures of the protein (Fig. 1b), indi-cating that a shorter protein SmSLK-S could also beexpressed in the parasite. To assess the presence of bothtranscripts in the parasite, adult worm RNA was analysedby Northern blotting using two different probes, one whichwas common to both SmSLK and SmSLK-S cDNAs(probe 1) and one which was complementary to thesequence deleted in SmSLK-S (probe 2). Results inFig. 2a confirmed the existence of large SmSLK transcripts(3.7 kb size) specifically recognised by probe 2 and showedwith probe 1 a more intense and broader band at about3.5–3.7 kb that was supposed to contain both SmSLKand SmSLK-S transcripts (which could not be discrimi-nated because of the small size of the deleted sequence).These results already indicated the existence of twoSmSLK variants in adult worms that were further con-firmed by real-time RT-PCR experiments in which we mea-sured the total amount of SmSLK and SmSLK-Stranscripts as well as selectively measuring the quantity ofSmSLK transcripts in different stages of the parasite.Tubulin gene levels were used as an internal standard. Data(Fig. 2b) showed that the SmSLK gene was differentiallyexpressed along the parasite life cycle with a maximum infemale worms and miracidia. Selective quantification ofSmSLK transcripts in each parasite stage allowed us todemonstrate that the shorter form of SmSLK-S transcriptswas largely represented in all the parasite stages studied,with a proportion varying from 45% (in female worms)up to 70% (in sporocysts) of the total transcripts of theSmSLK gene.

* * * * * SmSLK MLKSFFKKFISVEESGKKS--FSSTIERSVNPTDIWEIISELGDGAFGKVYKTHKRNTDLFAALKRVDFESEDELEDFMLEIDILTNFKHKNILTLHEVY 98LOK MAFANFRRILRLSTFEKRKSREYEHVRRDLDPNDVWEIVGELGDGAFGKVYKAKNKETGALAAAKVIETKSEEELEDYIVEIEILATCDHPYIVKLLGAY 100 Xplkk1 MAFANFRRILRLPNFEKKRLREYEHVRRDVDPNQVWEIIGELGDGAFGKVYKAKNWETGILAAAKVIETKNEEELEDYMVEIEILATCNHHFIVKLLGAF 100 mSLK MSFFNFRKIFKLGSEKKKK--QYEHVKRDLNPEEFWEIIGELGDGAFGKVYKAQNKETNVLAAAKVIDTKSEEELEDYMVEIDILASCDHPNIVKLLDAF 98

*** SmSLK IYESKLWIYLELCGGGALDSIMEALEKPLTEPQIRFVSREVLQGLEFLHEKLIIHRDMKAGNILLTLSNEVKLADFGVSAKLADEKQKRSTFIGTPYWMA 198 LOK YYDGKLWIMIEFCPGGAVDAIMLELDRGLTEPQIQVVCRQMLEALNFLHGKRIIHRDLKAGNVLMTLEGDIRLADFGVSAKNLKTLQKRDSFIGTPYWMA 200 Xplkk1 YWEGKLWIMIEFCPGGAVDAVMLELDRGLKEPEIKTICRQMLEALAYLHSMKIIHRDLKAGNVLLTLDGDIKLADFGVSAKNVKTLQRRDSFIGTPYWMA 200 mSLK YYENNLWILIEFCAGGAVDAVMLELERPLTESQIQVVCKQTLEALNYLHDNKIIHRDLKAGNILFTLDGDIKLADFGVSAKNTRTIQRRDSFIGTPYWMA 198

SmSLK PEVINCETFKDAPYNWKADIWSFGITLIELAQKRPPHNATNPTRVLLKILKSDPPTLSRPHLWSSKFKTFLGRTLQKDPNQRPECRDLLLDPFVSDV 295 LOK PEVVLCETMKDAPYDYKADIWSLGITLIEMAQIEPPHHELNPMRVLLKIAKSDPPTLLTPSKWSVEFRDFLKIALDKNPETRPSAAQLLQHPFVSRV 297 Xplkk1 PEVVMCETMKDAPYDYKADIWSLGITLIEMAQIEPPHHELNPMRVLLKIAKSEPPTLSSLSKWSPEFHSFLKTALDKNPETRPSAAQLLEHPFVKKA 297 mSLK PEVVMCETSKDRPYDYKADVWSLGITLIEMAEIEPPHHELNPMRVLLKIAKSEPPTLAQPSKWSSNFKDFLRKCLEKNVDARWTTSQLLQHPFVT-V 294

SmSLK TESDRKVIQILLCEVNADIIETVEDFD-PNEPIDEIDDNDLNPLILSDSTKLSIPVEMVCDDGDDDVDDHKNDVIGGEIDNSKNDHESNKNLKQINDNSG 394 LOK --TSNKALRELVAEAKAEVMEEIEDGREDGEEEDAVDAVPPLVNHTQDSANVTQPSLDSNKLLQDSSTPLPPSQPQEPVNGPCSQPSGDGPLQTTSPADG 395 Xplkk1 --SGNKPLRDLVAEAKAEVLDEIEEQGEAEEEEDS-DMLSPKTKGVSQSTHVEIG----------------KDIEKEQVGNGIKPHSATSPQNTDSQADN 378 AT1-46 ----------------------------------------------------------------------------------------------------

SmSLK DSGVSLGQTTSENQSDSNNKMNDHVIDEIADQLITDVITSETQSPSITSCVFESMNDIHSTIIKSPNTSSNSPATNITTTVVSNSTDKTEHVTKVNGNVD 494 LOK -----LSKNDNDLKVPVP-LRKSRPLSMDARIQMDEEKQIPDQD-------------------ENPSPAASKSQKANQSRPNSSALETLGGEALTNGGLE 470 Xplkk1 -----YSQRRNNEVKNCPENGRPDAVNRNPDIIILNPLSSNLEP-------------------KRNSTAESYRGEEHSSASSQRQRSAQSAELVPNGSFD 454 AT1-46 ----------------------------------------------------------------------------------------------------

SmSLK NRVRETIVHPKKQLNTSETTEHKSSVVNDILKDPPSSSPSATDFHDNNNKSKVVISPLKRQNSAYRTRTRTRRFVIDGQTITTTSKRIVNTNLEDKKFRE 594 LOK LPSSVTPSHSKRASDCSNLSTSESMDYGTSLS---------ADLSLNKETGSLSLKGSKLHN---KTLKRTRRFVVDGVEVSITTSKIIS---EDEKKDE 555 Xplkk1 SPTRYFTNWSKRDSDSGSNSASESMDISMNLS---------ADLSINKETGFLSHRENRLHK---KTLKRTRRFVVDGVEVSITTSKIIG---DDEKKDE 539 AT1-46 ------------------------MDYGTSLS---------ADLSLNKETGSLSLKGSKLHN---KTLKRTRRFVVDGVEVSITTSKIIS---EDEKKDE 61

SmSLK DEQIKRKAALRAFRILAKQEAHQTRELNERAQQQIDILENKMNAEFSTLTKTYDHKMEIAIRNYKLQMERLDKEFEVEMKRIRTETSKEERTFKDRLKNE 694 LOK EMRFLRRQELRELRLLQKEEHRNQTQLSSKHELQLEQMHKRFEQEINAKKKFYDVELENLERQQKQQVEKMEQDHSVRRK----EEAKRIRLEQDRDYAK 651 Xplkk1 EMRFLRRQELRELRLLQKEEHRHQAQLTSKHSFQLEQMSRRFEQEMNSKRKFYDTELETLERHQKQQIVWMEQEHAFRRR----DEAKHIKTEQERDHIK 635 AT1-46 EMRFLRRQELRELRLLQKEEHRNQTQLSTKHELQLEQMHRRFEQEINAKKKFYDVELENLERQQKQQVEKMEQDHSVRRR----EEAKRIRLEQDRDYAR 157

SmSLK IDTYDRAMRKAAKRDLNKYDHIIQSNSSLTSLKSNNSTTTTNTTTTTNNNNSSSLVSQRLTIFRENQESRMNSRIYDLHNEYNKRRNLIHSEYLNEIHTL 794 LOK FQEQLKQMKKEVKSEVEKLPRQQRKESMKQKMEEHSQKKQRLDRDFVAKQKEDLELAMRKLTTENRRE------ICDKERDCLSKKQELLRDREAALWEM 745 Xplkk1 FLEQLKLRKKELKAHVEKLPRQQRRETMKVQMDGFAHKKQTEEQQFVNRQKEDLNLAMRVIVLENRKE------IYNKEREFLNKKQQLLRDRESVIWEL 729 AT1-46 FQEQLKQMKKEVKNEVEKLPRQQRKESMKQKMEEHAQKKQLLDRDFVAKQKEDLELAMKKLTAENRRE------ICDKERDCLNKKQELLRDREAALWEM 251

SmSLK RLNFEEDKWRTEHRFLSMKHQSDRNRQLDLFMIKREQLTGRSELELTELKQTINMERSKLQAIHSIERKNCIKSMKSVHKRSILTLQRQSRLNTIQMN-E 893 LOK EEHQLQERHQLVKQQLKDQYFLQRHDLLRKHEKEREQMQRYNQRMMEQLKVRQQQEKARLPKIQRSDGETRMAMYKKSLHINGAGSASEQREKIKQFSQQ 845 Xplkk1 EERHLQERHQLVKQQLKDQYFLQRHELLRKHEKEQEQMQRYNQRMMEQLRLRQQQEKVRLPKNQKAEAKTRMTMFKKSLHISPSGSAAEQRDKIKQFSLQ 829 AT1-46 EEHQLQERHQLVKQQLKDQYFLQRHDLLRKHEKEREQMQRYNQRMMEQLKVRQQQEKARLPKIQRSDGKTRMAMYKKSLHINGAGSASEQREKVKQFSQQ 351

SmSLK AQKRMNEELELLESKQKAQTDELEKSIHFRLQELEQSTIEKRNALIDQETKRLQELDNRHQNEMRIYSESLPRIKALMKEQFAQEYKEDSFKLHNYHNYR 993 LOK EEKRQKAERLQQQQKHEHQMRDMVAQCESNMSELQQLQNEKCYLLVEHETQKLKALDESHNQSLKEWRDKLRPRKKALEEDLNQKKREQEMFFK------ 939 Xplkk1 EEKRQKAERLQQQQKHEHQLMEMLAECDCNVRDLLQMQNEKCHLLVEHETQKLKSLDEHHIQLIREWRENIRPRKKAFEDELELKKEAQEMFFR------ 923 AT1-46 EEKRQKAERLQQQQKHENQMRDMVAQCESNMNELQQLQNEKCHLLVEHETQKLKALDESHNQSLKEWRDKLRPRKKALEEDLNQKKREQEMFFR------ 445

SmSLK SYSPKSHKSRLPIPMTSLTNLRWGSLSNSNTKLNHITEPRSHRILTLFSSDLNNTKTGSTDSM 1056LOK --------------------------LSEEAEPRPTTPSKASNFFPYSSGDAS---------- 966 Xplkk1 --------------------------LNEEVAGDPFPSNKPTRFYSFSSPEAS---------- 950 AT1-46 --------------------------LSEEAETRPTTPNRASKFFPYSSGDAS---------- 472

0

0.2

0.4

0.6

0.8

1.0

200 400 600 800 1000

0.0

1200amino acid number

Prob

abilit

y of

coi

led-

coil

Ste20 signature

Alternative insert

Fig. 1. Amino-acid sequence and structure analysis of SmSLK. (a) Alignment of SmSLK kinase domain (residues 1–295) with that of the Ste20-relatedkinases, mouse LOK (Kuramochi et al., 1997; BAA24073, residues 1–297), mSLK (Sabourin and Rudnicki, 1999; NP_033315, residues 1–294), and xPlkk1from Xenopus (Qian et al., 1998; AAC95157, residues 1–297) by the Clustal W method. Shaded areas correspond to residues which are identical (in black)or similar (in grey) in at least 75% of the aligned sequences. Conserved residues in ATP and cofactor (Mg++) binding sites are indicated by asterisks andthe Ste20 signature is overlined. (b) Alignment of the SmSLK non-catalytic region (residues 296–1056) with that of LOK (residues 298–966), xPlkk1(residues 298–950) and with the rat protein AT1-46 (Schaar et al., 1996; AAC52648, residues 1–472). The sequence deleted in the short form of SmSLK(SmSLK-S, see Fig. 2) is overlined. (c) Analysis of the coiled-coil region of SmSLK. The probability of forming a coiled-coil structure was calculated foreach residue with a window of 28 amino acids by the method of Lupas et al. (1991).


Fig. 2. Expression and quantification of two different transcripts ofSmSLK. (a) Northern blot analysis of total RNA from Schistosoma

mansoni adult worms. Transcripts of approximately 3.7 kb were detectedusing probe 1 recognising both SmSLK isoforms (SmSLK + SmSLK-S)or probe 2 selectively recognising SmSLK. (b) Quantification of SmSLKtranscripts in the different developmental stages of the parasite. Quanti-tative amplifications of total SmSLK and SmSLK-S cDNAs as well as ofcDNAs specifically encoding SmSLK were performed in triplicate.Tubulin gene levels served as internal standards in each parasite stage.For graphical representation of quantitative PCR data, cycle thresholds(DCt values) obtained for total (SmSLK + SmSLK-S) transcripts in thedifferent stages were deducted from the DCt value obtained for male adultworm transcript levels. Values were normalised as fold-difference relativeto male worms using the delta–delta Ct (DDCt) method (Livak andSchmittgen, 2001). For each stage, the DCt value obtained for SmSLK-specific transcript levels was deducted from that obtained for total(SmSLK + SmSLK-S) transcripts. Using the same method, the respectiveproportion of SmSLK (hatched area) and SmSLK-S cDNAs wascalculated for each parasite stage.


3.3. SmSLK gene organisation

In order to confirm the presence of alternative exon(s) inthe SmSlk gene, the assembly of its complete structure(Fig. 3 and Supplementary Table 1) was performed using

Fig. 3. Exon–intron structure of the SmSLK gene. Exons (boxes) and definedprotein sequence are given in Supplementary Table 1. Introns of unknown sizeto the SmSLK-S product is indicated below the gene structure. Start (ATG) andexons 1–7) as well as coiled-coil CC1 (exons 11–14) and CC2 (exons 14–18) re

the S. mansoni genomic sequences available from the gen-ome sequencing project (see Section 2). Only one copy ofthe gene was found. All intron–exon junctions were verifiedby direct sequence alignment and all intron junctionsequences correspond to the consensus 5 0 GT–AG 3 0 struc-ture invariably found in schistosome genes (RaymondPierce, unpublished data). The gene is composed of 18exons with two small (35 and 33 bp) introns at the 5 0 endof the gene. The other defined introns are all >1500 bp(up to 6663 bp), and the sizes of seven introns were notdetermined due to the presence of gaps in the genomicsequence; the overall size of the gene is >43,760 bp. Thegene structure confirms that the two SmSLK isoforms cor-respond to splicing variants; SmSLK cDNA contains exon9, whereas SmSLK-S lacks this exon. The alternative exon9 encodes a peptide located between the kinase domain andthe first coiled-coil domain. Kinase (exons 1–7) and coiled-coil domains (exons 11–14 and 14–18) are common to bothisoforms (Fig. 3).

3.4. Expression of SmSLK proteins and demonstration oftheir kinase activity

To study the kinase activity of SmSLK proteins, wecloned the full-length sequences of SmSLK and SmSLK-Sin-frame into the pcDNA3.1-V5/His expression vector.HEK293T were transiently transfected by SmSLK plasmidsas well as by the myc-tagged mouse SLK (Sabourin andRudnicki, 1999) as a positive control of expression andkinase activity. Kinase assays were performed on cellextracts immunoprecipitated, respectively, by anti-V5 oranti-myc antibodies using MBP as a substrate. Results inFig. 4a demonstrated that both SmSLK and SmSLK-Swere able to phosphorylate the exogenous MBP substratesimilarly to the mouse kinase mSLK, a 20 kDa radioactiveband corresponding to phospho-MBP being detected in thethree kinase reactions but not with lysates from cells trans-fected with empty plasmids. A purified fraction of the activekinase v-Abl was used as a positive control for the detectionof phospho-MBP. In SLK kinase reactions, labelled bandswere also detected at the top of the gel, which probably rep-resent autophosphorylated forms of SmSLK-S, SmSLK

introns (solid line) are to scale. Sizes of exons and amino-acid limits in theare designated by an interrupted line. The alternative splicing event leading

stop (TGA) codons are marked by arrows. Kinase domain (encompassinggions are indicated.

Fig. 4. Kinase activity of recombinant SmSLK proteins expressed in HEKcells. (a) Lysates from cells transfected by SmSLK-S or SmSLK-pcDNAV5 or by empty pcDNA V5 plasmids (control) were immunoprecipitatedby anti-V5 antibodies. Transfection by mSLK-pcDNA myc plasmid wasused as a positive control of protein expression and activity. Kinasereactions were performed on immunoprecipitates in the presence of[c-32P]ATP and MBP substrate, then analysed by SDS–PAGE and gelautoradiography. A purified fraction of active v-Abl protein tyrosinekinase was used as a positive control in kinase assays. The 20 kDa labelledband corresponds to phosphorylated MBP. Autophosphorylated forms ofmSLK (110 kDa) and SmSLK/SmSLK-S (120 kDa) could be detected aswell as autophosphorylated v-Abl proteins (50 kDa). (b) Similar assayswere performed following transfection of HEK cells with dead kinasemutants of SmSLK-S (SmSLK-SDK) and SmSLK (SmSLKDK). Noradioactive MBP was detected with dead kinase mutants. Immunoblotsof cell extracts revealed with anti-V5 or anti-myc antibodies confirmed theexpression of the different V5-tagged SmSLK and myc-tagged mSLK inHEK transfected cells.

Fig. 5. SmSLK activates the JNK pathway in Xenopus oocytes. (a)Immunoblot analysis of soluble extracts from oocytes microinjected withSmSLK or SmSLKDK cRNA and incubated for 48 h. Blots wereincubated with anti-V5, anti-c Jun N-terminal kinase (anti-JNK) or


and mSLK, respectively, (Fig. 4a). Further experiments per-formed using dead-kinase mutants of SmSLK proteins(SmSLK-SDK and SmSLKDK) demonstrated that a singlemutation in the Mg2+ binding site (DFG/DNA motif) inSmSLK proteins was sufficient to inhibit the kinase activityof parasite proteins, thus confirming the specificity of kinasereactions (Fig. 4b). In all these assays, Western blot analysisof HEK293T cell homogenates with anti-tag antibodiesassessed the expression of both parasite and mouse proteinsin transfected cells (Fig. 4b).

anti-phosphoactive JNK (anti-JNK-P) antibodies and HRP-labelledsecondary antibodies were detected using the ECL Western blottingdetection system. A chemoluminescent signal was detected with anti-JNK-P antibodies in oocytes injected with the wild type SmSLK but not withthe dead kinase SmSLKDK. (b) Time-course detection of phosphorylatedJNK proteins in oocytes expressing SmSLK-S or SmSLK proteins for 6,24 or 48 h.

3.5. SmSLK activates c-Jun amino-terminal kinase 1

(JNK1)

Biochemical evidence has been obtained indicating thatmouse SLK over-expression in cultured fibroblasts acti-

vates the MAPK, JNK1 (Sabourin and Rudnicki, 1999).In this work, we have investigated the potential effect ofSmSLK proteins on JNK1 phosphorylation and activationin two different cellular systems. Xenopus oocytes, whichare frequently used to produce heterologous proteins, havealready been shown to express with efficacy S. mansonikinases involved in MAPK signalling (Vicogne et al.,2004). Results presented in Fig. 5a showed the efficienttranslation of SmSLK cRNA, in vitro-transcribed fromSmSLK- pcDNA V5 plasmids, in oocytes. Fourty-eighthours after injection, the presence of V5-tagged SmSLKproteins could be detected in homogenates of injectedoocytes by Western blot analysis. Using anti-phospho-JNK antibodies, we could also demonstrate that JNK ofXenopus was phosphorylated in SmSLK-expressingoocytes. When oocytes were injected with SmSLKDK

pcDNA V5 plasmids, the presence of phospho-JNK wasnot detected, whereas similar levels of JNK proteins wererecognised by anti-JNK antibodies in both oocyte prepara-tions. These results indicated that JNK phosphorylationwas dependent on SmSLK kinase activity. Experiments inwhich SmSLK-S-cRNAwas injected into oocytes demon-strated that the exon 9-deleted variant of SmSLK couldalso induce the phosphorylation of JNK 48 h afterinjection but with low efficacy compared with SmSLK.Time-course studies also showed that a period of 48 hwas necessary for maximal protein synthesis (results notshown) and to observe the phosphorylation of JNK pro-teins in the presence of SmSLK and SmSLK-S (Fig. 5b).Distinct experiments to investigate JNK/MAPK activation

Fig. 6. SmSLK activates c-Jun amino-terminal kinase 1 (JNK-1) inHEK293T cells. Lysates from cells co-transfected with a vector encodingFLAG-tagged JNK-1 together with an empty pcDNA vector (control),SmSLK or SmSLK-S-pcDNA V5 or mSLK-pcDNA myc plasmids wereimmunoprecipitated by anti-FLAG antibodies. Kinase reactions wereperformed on anti-FLAG immunoprecipitates in the presence of[c-32P]ATP and purified GST-jun protein as a specific substrate, thenanalysed by SDS–PAGE and gel autoradiography. The position of theGST-jun labelled band is indicated. Immunoblots of cell extracts withanti-FLAG, anti-V5 or anti-myc antibodies confirmed the expression ofJNK1 in HEK transfected cells and that of the V5-tagged SmSLK andmyc-tagged mSLK, respectively.


were performed using mammalian HEK293T cells co-transfected with SmSLK plasmids together with a plasmidencoding the JNK-1 kinase (Paumelle et al., 2000). In theseassays, we measured the activation of JNK-1 by its capac-ity to phosphorylate, in vitro, its specific substrate c-jun.The autoradiogram in Fig. 6 indicates that JNK-1 imuno-precipitated from cells transfected by SmSLK-containingplasmids was able to phosphorylate GST-jun proteins.JNK1 activity appeared to be lower in cells transfectedby SmSLK-S plasmids, corroborating the results obtainedwith Xenopus oocytes. In these experiments, mouse SLKwas also shown to activate JNK-1 (Fig. 6), confirming datapreviously published (Sabourin and Rudnicki, 1999).Immunoblotting of cell lysates showed that FLAG-taggedJNK-1 as well as V5 and myc-tagged SLK proteins wereexpressed in transfected cells.

3.6. SmSLK is expressed in the parasite tegument

In situ immunolocalisation was performed on adultmale sections using immunoglobulins produced against arecombinant polypeptide corresponding to the non-con-served protein domain of SmSLK (amino acids 289–612)and shown to recognise a unique protein of about130 kDa in adult worm extracts in Western blot analysis

(results not shown). Data in Fig. 7 indicated that SmSLKproteins were expressed abundantly in the parasite tegu-ment, suggesting that they could participate in signallingduring host–parasite relationships. Fluorescent labellingwas also observed in the parenchyma of the parasite. Thissuggested that SmSLK proteins were expressed ubiqui-tously in the adult worm and could play, as do otherSLK proteins, multiple roles in kinase-dependent pathwaysinvolved in schistosome biology.

4. Discussion

Although a variety of signalling proteins have been iden-tified in S. mansoni, including surface receptor and cyto-solic kinases (Dissous et al., 2006), little is known aboutsignal transduction mechanisms in these parasites. In S.

japonicum, in silico analysis of the MAPK signalling path-ways has recently confirmed their evolutionary conserva-tion, allowing the identification of at least 60 putativemembers of the MAPK signalling pathways already knownin organisms ranging from yeast to mammals. Such con-served pathways in schistosomes are supposed to beinvolved in the regulation of the complex parasite life cycleas well as in host–parasite interactions (Wang et al., 2006).

In this paper, we describe the characterisation of a novelSer/Thr kinase of S. mansoni, homologous to the membersof the Ste20 kinase group. Ste20p was originally identifiedas a yeast MAPK4 involved in the Fus3 mating pathway ofS. cerevisiae. Homologs of Ste20p have been found inmammals and diverse organisms. These constitute a largegroup of protein kinases, including at least 30 found inhumans (Pombo et al., 2007). Ste20-like kinases are charac-terised by a conserved kinase domain and a variable non-catalytic domain that mediates interaction with a largepanel of signalling molecules regulating cell growth, differ-entiation and death, as well as cytoskeletal rearrangements.

The catalytic domain of SmSLK contains the 11 con-served subdomains of Ser/Thr kinases required for phos-phorylation activity (Hanks and Hunter, 1995). Wedemonstrated that purified recombinant SmSLK was anactive kinase able to phosphorylate the exogenous MBPsubstrate and it was also able to autophosphorylate(Fig. 4a). The presence in subdomain VIII of the Ste20 sig-nature motif also confirmed that SmSLK belongs to theSte20 kinase group.

The Ste20-like group is divided into PAK and GCKfamilies, differing in the location of their kinase domainwhich is positioned at the C-terminus of PAK proteinsand at the N-terminus of GCK proteins. Based on thesefeatures the parasite protein SmSLK, composed of a con-served N-terminal kinase domain and a large C-terminaldomain, could be classified as a member of the GCK fam-ily. Results of homology research further confirmed thesimilarity of SmSLK with previously characterised GCKmembers. Sequence alignment of the non-catalytic domainof SmSLK revealed similarity with the C-termini of LOKproteins, which have already been shown to be identical

Fig. 7. Immunolocalisation of SmSLK proteins in longitudinal sections of male adult worms of Schistosoma mansoni. Sections were labelled with SmSLKimmune mouse serum and with (Fab 0)2 goat anti-mouse antibodies conjugated to Alexa 488 fluorochrome (A,B). SmSLK was detected in the tegument(T) as well as in the parenchyma (P) of male worms. Negative control (C) was incubated with mouse pre-immune serum.


to 60–70% of the rat brain AT1-46 protein (Schaar et al.,1996). Those sequences common to LOK proteins andAT1-46 were predicted to adopt a-helical and coiled-coilstructural conformation and constitute important regula-tory domains for protein-protein interactions. The presencein SmSLK of similar coiled-coil structures was confirmedby sequence analysis using the COILS program (Lupaset al., 1991). Similarities were further found betweenSmSLK and mouse SLK sequences but they were restrictedto the AT1-46-like coiled-coil motifs also contained inmouse SLK. Indeed, mouse SLK exhibits a complex C-ter-minal region including additional microtubule and nuclear-associated proteins (M-NAP)-related sequences in itscentral part, that are probably involved in interactions withother protein partners. M-NAP sequences are absent fromSmSLK and LOK proteins. All these data confirm thatSmSLK is a new member of the GCK family, likely belong-ing, as do LOK and SLK proteins, to the GCK-V subfam-ily (Dan et al., 2001).

The assembly of the SmSLK gene structure, carried outusing the S. mansoni genomic sequences available from thegenome sequencing project, indicated that SmSLK wascomposed of 18 exons. The first seven (1–7) exons encodethe catalytic domain and the last eight (11–18) exonsencompass the coiled-coil regulatory domains. Sequenceanalysis of exons 8, 9 and 10 did not reveal any structurallyconserved motifs. One particularity for the SmSLK genewas the generation of two transcripts, corresponding tosplicing variants of exon 9, the expression of which couldbe confirmed by Northern blotting and selective PCRamplification (Fig. 2). SmSLK cDNA contains exon 9whereas, SmSLK-S lacks this exon. According to the liter-

ature, the existence of splicing variants in GCK membersdoes not appear to be a general feature. However, anobservation similar to that made for SmSLK has beenreported for KFC (Kinase From Chicken), a member ofthe Ste20 kinase group identified in chicken embryo fibro-blasts, that is also expressed as a splicing variant deleted fora serine-rich sequence located, as in SmSLK, between theN-terminal catalytic domain and the C-terminal coiled-coilregulatory domains. The authors demonstrated that KFCand KFC-S (spliced form) proteins not only differ in struc-ture, but also in biological properties, particularly in theirpotential to alter the growth of chicken fibroblasts (Yusteinet al., 2000) which was higher for KFC than for KFC-S.

Protein Ser/Thr kinases related to GCK have emergedas important players in the regulation of stress-activatedMAPK core signalling pathways. The large family ofGCKs has been subdivided into two broad groups, groupI and group II, according to their functional properties(Kyriakis, 1999). Group I GCKs include in their C-termi-nal domains several polyproline consensus binding sitesfor proteins containing Src homology (SH)-3 domainswhich are not found in group II GCKs. As is the case forLOK, Stk10 and mouse SLK, SmSLK does not containSH3-binding sites and can be structurally related to theGCKs of group II.

Although group I GCKs have been largely demon-strated to participate in the activation of the stress-acti-vated protein kinase (SAPK/JNK) pathway (Kyriakis,1999), the role of group II GCKs in this pathway is contro-versial. Indeed, mouse LOK proteins activate none of theknown MAPK kinase cascades (Kuramochi et al., 1997)whereas mouse SLK has been shown to activate JNK-1


and to induce apoptosis (Sabourin and Rudnicki, 1999) inmammalian cells. We demonstrated that kinase activity ofJNK-1 molecules could be efficiently induced in mouseSLK-transfected cells as well as in SmSLK-transfectedcells. In both cases, JNK-1 molecules could phosphorylatetheir specific c-jun substrate, confirming the previousresults obtained for mouse SLK (Sabourin and Rudnicki,1999) and demonstrating a similar role for SmSLK in acti-vation of the SAPK/JNK pathway. Additional studiesusing heterologous expression of SmSLK proteins in Xeno-

pus oocytes confirmed the ability of SmSLK to induce thephosphorylation of JNK, a process necessarily required forkinase activation. Phosphorylation of the MAPK wasdependent on the kinase activity of SmSLK, as shown bythe inefficacy of a dead kinase mutant SmSLKDK to phos-phorylate oocyte JNK proteins. Taken together, theseresults strongly supported the participation of SmSLK inactivation of the SAPK/JNK pathway. Moreover, itshould be mentioned that in both HEK cells and Xenopus

oocytes, SmSLK-S was less efficient than SmSLK in phos-phorylating and activating JNK molecules, raising the pos-sibility that, as was already shown in the case of KFCproteins (Yustein et al., 2000), the peptide insert encodedby exon 9 in SmSLK is important for SmSLK biologicalactivity.

RT-PCR experiments have demonstrated the expressionof the SmSLK kinase in the different life stages of the schis-tosome, suggesting a ubiquitous role for SmSLK during theparasite life cycle. However, quantitative RT-PCR datarevealed a differential expression of the SmSLK gene in thevarious stages. They showed that transcription was moreactive in miracidia and in female worms and that SmSLK-S transcripts were present in a large proportion (from 45 to70%) in each stage. In miracidia, we could assume that thelarge quantity of SmSLK transcripts reflects the abundanceof various transcripts generally observed in this free-livingparasite stage (Khayath et al., 2006). Indeed, miracidia seemto stock a large panel of mRNA coding for proteins that willbe quickly required upon transformation into sporocysts inthe intermediate host.

Immunofluorescence studies have indicated thatSmSLK proteins were dispersed in the parenchyma of theworm, suggesting multiple roles in kinase-dependent path-ways involved in the biology of the schistosome. Moreover,SmSLK has been found to be abundantly expressed in theparasite tegument, and this result could be in agreementwith its potential participation in MAPK (SAPK/JNK)signalling and the regulation of host–parasite relationships.Concerning the abundance of SmSLK transcripts in femaleworms, it is tempting to speculate that SmSLK might beinvolved in essential functions of development and repro-duction and might therefore represent a potential candi-date for new therapeutic strategies aimed at reducingworm fertility and schistosomiasis pathology. In this con-text, it is of interest to note that important structuralhomologies exist between SLK or LOK proteins and thepolo-like kinase kinase 1 (xPlkk1) of Xenopus (Qian

et al., 1998), another Ste20-like kinase belonging to thesame group II of GCKs and that phosphorylates the Xeno-

pus polo-like kinase 1 (xPlk1). Plk1 molecules are essentialregulators of mitosis in all eukaryotic cells and for this rea-son are now considered potential targets for cancer therapy(Strebhardt and Ullrich, 2006). The recent demonstrationthat both mouse SLK (O’Reilly et al., 2005) and humanStk10 (Walter et al., 2003) proteins were also able to acti-vate Plk1 in mammalian cells, raises the question of a pos-sible polo-like kinase kinase activity of SmSLK. Work isnow in progress to examine the expression of SmSLK infemale tissues and particularly in reproductive organs,and its potential implication in sexual maturation, the mul-tiplication of germinal cells and egg production.

Acknowledgements

This work was supported by the Institut National de laSante et de la Recherche Medicale, the Centre National dela Recherche Scientifique, the Institut Pasteur de Lille andthe Universite de Lille 2.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.ijpara.2007.06.001.

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Molecular cloning and characterisation of SmSLK, a novel Ste20-like kinase in Schistosoma mansoni