Characterization of the Src/Abl Hybrid Kinase SmTK6 of Schistosoma mansoni*

Background: SmTK6 was identified as interaction partner of SmTK4. Results: SmTK6 is a Src/Abl hybrid kinase and interacts also with the uncommon SmVKR1 and SmTK3. Conclusion: SmTK6 is suggested to be part of a complex of receptors, Syk and Src kinases, which are involved in gonad development. Significance: SmTK6 represents an Abl kinase progenitor, for which a function in reproduction could be assigned. Cellular protein-tyrosine kinases play key roles in signal transduction processes in eukaryotes. SmTK4 was the first Syk kinase identified in a parasite and found to be tissue-specifically transcribed in the gonads of adult Schistosoma mansoni. Functional analyses confirmed its role in oogenesis and spermatogenesis. As an SmTK4 upstream binding partner, the cellular protein-tyrosine kinase SmTK6 was isolated from a yeast two-hybrid library. Phylogenetic analyses performed in this study confirmed the first suggestions of a hybrid character of SmTK6. Biochemical studies made in Xenopus oocytes using inhibitors against Src (herbimycin A) and Abl (imatinib) kinases exhibited a biochemical inhibition profile of SmTK6, which was intermediate of Src and Abl kinases. As SmTK6 upstream interaction partners, we identified among others the known Src kinase SmTK3 and the Venus kinase receptor SmVKR1 of S. mansoni by yeast two-hybrid analyses, all of which co-localized in the gonads. Co-immunoprecipitation experiments confirmed interactions between SmTK6 and SmTK3 or SmVKR1. In Xenopus oocytes, it was finally shown that SmVKR1 but also SmTK3 were able to activate SmTK6 enzymatic activity indicating its functions in a receptor tyrosine kinase signal transduction cascade. These results not only demonstrate an intermediate but Src-biased profile of the unusual kinase SmTK6. They also strongly substantiate previous indications for a kinase complex, consisting of a receptor tyrosine kinase, Syk and Src kinases, which has been hypothesized to be involved in proliferation and differentiation processes in the gonads of schistosomes.


SUMMARY
Cellular protein tyrosine kinases (CTKs) play key roles in signal transduction processes in eukaryotes. SmTK4 was the first Syk kinase identified in a parasite and found to be tissue-specifically transcribed in the gonads of adult Schistosoma mansoni. Functional analyses confirmed its role in oogenesis and spermatogenesis. As an SmTK4 upstream binding partner the CTK SmTK6 was isolated from a yeast two-hybrid library. Phylogenetic analyses performed in the present study confirmed first suggestions of a hybrid character of SmTK6. Biochemical studies made in Xenopus oocytes using inhibitors against Src (Herbimycin A) and Abl (Imatinib) kinases exhibited a biochemical inhibition profile of SmTK6, which was intermediate of Src and Abl kinases. As SmTK6 upstream interaction partners we identified among others the known Src kinase SmTK3 and the Venus Kinase Receptor SmVKR1 of S. mansoni by yeast two-hybrid (YTH) analyses, all of which co-localized in the gonads. Co-immunoprecipitation experiments confirmed interactions between SmTK6 and SmTK3 or SmVKR1. In Xenopus oocytes it was finally shown that SmVKR1 but also SmTK3 were able to activate SmTK6 enzymatic activity indicating its functions in a RTK signal transduction cascade. These results not only demonstrate an intermediate but Src-biased profile of the unusual kinase SmTK6. They also strongly substantiate previous indications for a kinase complex, consisting of a RTK, Syk-and Src-kinases, which has been hypothesized to be involved in proliferation and differentiation processes in the gonads of schistosomes.
As essential members of signal transduction cascades cellular protein tyrosine kinases (CTKs) are involved in processes regulating cytoskeletal reorganization, migration, proliferation, development and differentiation, metabolic homeostasis, transcriptional activation, neural transmission, aging and survival (1). CTKs forward incoming signals from transmembrane receptors to binding partners acting downstream in a signaling hierarchy (2,3). Depending on their function and state of activation, CTKs can be either located in the cytoplasm, be attached to membranes, or occur in the nucleus (4). According to their structural features, CTKs were classified as distinct families: Src, Abl, Syk, Jak, Fak, Fes/Fer, Csk and Btk (5). Among these, Src and Abl kinases have Src-homology (SH3, SH2) domains and a catalytic tyrosine kinase (TK) domain in the same linear order. Src kinases possess two conserved regulatory tyrosine (Tyr, Y) phosphorylation sites (1,5). Phosphorylation of Tyr 527 within the C-terminus is important for the inactive conformation of Src, mediated by intramolecular binding to its SH2 domain, whereas phosphorylation of Tyr 416, an autophosphorylation site within the activation loop of the TK domain, leads to the stimulation of kinase activity. SH2 and SH3 domains have been implicated in the negative regulation of Src activity, but both also contribute to the binding of partners acting upstream (via SH2) or downstream (via SH3) of Src in signaling cascades, if this kinase becomes activated (5). Regulation of Abl kinases differs as they lack Tyr 527 within the C-terminus, which has no functional role in the control of Abl-kinase activity. Instead the inactive conformation is formed by intramolecular binding of a short N-terminal cap peptide, supported by a myristoyl group attached to the amino-terminal glycine, to the TK domain. Downstream of the cap peptide are SH3 and SH2 domains contributing by interaction with the TK domain to the "locked" inactive state (6). Further features of Abl kinases from higher eukaryotes are the presence of a nuclear localization sequence, and an elongated C-terminal region containing varying sets of binding domains for actins, microtubules, or DNA (6).
Schistosomes are helminth parasites causing schistosomiasis, one of the most prevalent parasitic diseases for human and animals worldwide (7,8). Its pathology is directly associated with egg production of mature adult worms. Eggs cause granuloma formation and inflammatory processes, which interfere with organ function. The course of disease can be fatal affecting millions of people (9). As the only class within the trematodes, schistosomes have evolved separate sexes. Furthermore, by a continuous pairing contact with the male, proliferation and differentiation processes are initiated in female gonads (10)(11)(12). This is the prerequisite for the synthesis of composite eggs consisting of oocytes originating from the ovary and vitelline cells delivered from the vitellarium. During the last years, first molecules have been characterized that are involved in regulating mitogenic activity and differentiation in female gonads. Among these are the Src kinase SmTK3 and the Syk kinase SmTK4 (13,14). SmTK3 contains all Src-typical features, and expression products were localized in vitelline cells, oocytes, and spermatocytes suggesting a role in reproductive activity (15). Studies with the Src kinase-specific inhibitor Herbimycin A provided strong evidence for a role of SmTK3 in the control of mitotic activity and egg production of adult schistosomes in vitro (16). Characterized by its tandem SH2-domains SmTK4 is a typical Syk kinase. SmTK4 transcripts were found in spermatocytes and oocytes, but not in vitelline cells (17). Using the Syk kinase-specific inhibitor Piceatannol and RNAi knock-down approaches in adult worms in vitro demonstrated a decisive role of SmTK4 in oogenesis and spermatogenesis (13). As SmTK4 upstream interaction partner SmTK6 was identified and co-localized in the reproductive organs. Coimmunoprecipitation experiments confirmed direct interactions between both kinases (13). First data base analyses comparing SmTK6 with two recently detected Abl kinases from schistosomes suggested that SmTK6 may represent a Src-/Abl-like hybrid kinase (18).
In this study we provide functional evidence for the intermediate Src/Abl kinase characteristic of SmTK6 by gene-structure and phylogenetic analyses, but also by inhibitor studies. Furthermore, we identified upstream bindingpartners in S. mansoni such as SmTK3, SmVKR1, a Drosophila Discs-large homolog (DLG), and a new transmembrane mucin. Transcripts of all these genes co-localized in the reproductive organs. Following coimmunoprecipitation experiments which confirmed SmTK6-SmTK3 as well as SmTK6-SmVKR1 interactions, germinal vesicle breakdown (GVBD) assays in Xenopus oocytes finally demonstrated that SmTK6 can be activated by SmVKR1 or SmTK3. These results reinforce previous suggestions of a multi-kinase complex in the gonads of schistosomes consisting of the Syk kinase SmTK4, the Src kinase SmTK3, and the RTK SmVKR1, in which the unusual Src/Abl-like kinase SmTK6 is a novel player.

EXPERIMENTAL PROCEDURES
Parasite stock-Adult and larval schistosome stages originated from a Liberian isolate of S. mansoni (19), which was maintained in snails (Biomphalaria glabrata) and Syrian hamsters (Mesocricetus auratus). Adult worms were obtained by hepatoportal perfusion at 42 -49 SmTK6 of S. mansoni days post-infection. Experiments with hamsters were performed in accordance with the European regulations (ETS No 123; revised Appendix A) and were approved by the Regional Council Giessen, Germany.
Library screening was performed according to the user manual (Yeast protocols handbook, Clontech). In short, yeast cells (strain Y187) were transformed with the bait plasmid SmTK6-SH2 pBridge by lithium acetate. Bait-expressing Y187 cells were mated with library-containing AH109 cells. The first selection of diploid yeast cells was carried out on synthetic dropout medium lacking tryptophan, leucine, and histidine (Trp -/Leu -/His -). To enhance the selection pressure on clones with interacting proteins, colonies were plated onto synthetic dropout medium additionally lacking adenine (Trp -/Leu -/ His -/Ade -). For further selection, β-galactosidase (β-gal) colony filter assays were performed using X-Gal (5-bromo-4-chloro-3-indolyl-β-Dgalactopyranoside) as substrate according to the manufacturer"s instructions (Yeast protocols handbook, Clontech). From positively tested yeast clones, plasmid DNA was isolated using cell disruption by vortexing with glass beads (Sigma) followed by plasmid preparation (peqGOLD Plasmid Mini Kit, PeqLab). Plasmid DNA was transformed into heat shock-competent Escherichia coli cells (DH5α) followed by selection on LB-plates containing ampicillin (100 µg/µl). To differentiate bacterial colonies containing bait plasmids from those containing prey plasmids, colony PCRs with pGADT7specific primers were performed. Prey plasmids from PCR-positive bacterial clones were isolated and sequenced commercially (LGC Genomics, Berlin). To confirm protein-protein interactions, the yeast strain AH109 was transformed with appropriate prey plasmids together with the bait plasmid, and the selection procedures were repeated. For quantification of relative interaction strengths, β-gal liquid assays with ONPG (o-nitrophenol-galactopyranoside) as substrate were performed according to conven-tional protocols (Yeast protocols handbook, Clontech).
Full-length (fl) SmTK6 and SmTK3 were cloned into pcDNA 3.1. SmTK6 plasmids were linearized by PmeI or by EcoRI (position nt 920) to produce cRNA encoding full length SmTK6 (SmTK6-fl) or only its N-terminal part containing SH3SH2 domains without the TK domain (SmTK6-SH3SH2), respectively.
Dead-kinase variants of full length SmTK6 and SmTK3 (SmTK6-fl-ko and SmTK3-fl-ko) were generated by changing the Mg ++ binding motif DFG present in their kinase domain into a DNA motif, as previously described (22). SmTK6-SmTK3 interaction studies were performed by co-injection of cRNA produced from the different versions of SmTK6 and SmTK3 plasmids.
Transcriptional analyses-To confirm the transcription of the bait vector transgene SmTK6-SH2 in transformed yeast cells, a 5 ml over-night culture of an appropriate yeast clone was centrifuged. The pellet was washed twice with PBS and frozen in liquid nitrogen. Cells were disrupted by three freeze/thaw cycles (liquid nitrogen, 37°C water bath), 1 ml TriFast (PeqLab) was added to the lysate, and total RNA was extracted according to the manufacturer"s instructions. The synthesis of cDNA was done with 90 ng total RNA, a primer specific for the SH2 domain of SmTK6 (SmTK6-SH2-3"), and Sensiscript reverse transcriptase (Qiagen). RT-PCR analyses were performed using 1/4 of the cDNA as template, FIREPol Taq-polymerase (Solis BioDyne), and the primer combination SmTK6-SH2-5" + SmTK6-SH2-3".
To investigate the stage-specific transcription of SmTK6 in S. mansoni, RT-PCR analyses were performed with total RNA of adult or larval worms extracted by TriFast (PeqLab) following the manufacturer"s instructions.
In situ hybridizations were done as described elsewhere in detail (20). In short, adult worm pairs were fixed in Bouin"s solution (picric acid/acetic acid/formaldehyde; 15:1:5) before embedding in paraplast (Histowax, Reichert-Jung). Sections of 5 µm were generated and incubated in xylol to remove the paraplast. Following re-hydration, proteins were removed by proteinase K treatment (final concentration 1 µg/ml), and the sections dehydrated. For hybridization, in vitro-generated transcripts were labeled with digoxigenin following the manufacturer"s instructions (Roche). Labeled sense and antisense transcripts of SmDLG (406 bp; position bp 974-1379) or SmVKR1 (322 bp; position 3938 -4260) were size-controlled by gel electrophoresis. To prove their quality, transcript blots were made to confirm digoxigenin incorporation by alkaline phosphataseconjugated anti-digoxigenin antibodies, naphthol-AS-phosphate, and Fast Red TR (Sigma). All in situ hybridizations were performed for 16 h at 42°C. Sections were stringently washed up to 0.5x SSC, and detection was achieved as described for transcript blots.
In silico analyses-The following public domain tools were used for sequence analyses: NCBI-BLAST

RESULTS
The SmTK6 full-length cDNA sequence reveals Src and Abl characteristics-By Y2H cDNA-library screening using the tandem SH2domain of SmTK4 as bait, the known Src kinase SmTK3 (15; accession number CAE51198), and the novel kinase SmTK6 (accession number FN397679) were identified as potential upstream binding partners (13). Comparative analyses of the relative binding strengths by β-gal liquid assays showed a weak interaction between SmTK4 and SmTK3, and a strong interaction between SmTK4 and SmTK6. The binding potential of SmTK4 and SmTK6 was finally confirmed by co-immunoprecipitation (13).
The SmTK6 cDNA has a length of 1698 bp coding for a protein of 565 aa. Domain structures such as SH3 (aa 115-172), SH2 (aa 179-268), and a catalytic TK domain (aa 297-551) are present (supplementary data 1) indicating the similarity of SmTK6 to Srcor Abl-kinases. A SH4 domain functionally important for intracellular membrane attachment via myristoylation may not exist. Although a typical glycine (G) occurs at position 2 (MGICLC), it is not embedded in a consensus sequence characteristic for myristoylation (MGXXXS/T) (28). Since two typical cysteine (C) residues were detected at positions 4 and 6, palmitylation may be possible instead (29). Compared to other Src-kinases, SmTK6 possesses only one of the typical Tyr residues with regulatory function (Y562). It is positioned within the C-terminus close to the TK domain at a conserved position compared to Y527 of human c-Src (Fig. 1A, supplementary data 1). Tyr 416 of human Src, which occurs in the Cterminal half of the TK domain, is missing in SmTK6. Instead a serine (Ser, S) residue occurs at the corresponding position 447 (supplementary data 1). Within the catalytic TK domain further conserved regions exist, of which two distinguish between serine/threonine kinases and TKs (30). SmTK6 possesses the TK-specific D-L-A-A-R-N/D-L-R-A-A-N motif of subdomain I (aa 418-423), and the P-I/V-K/R-W-T/M-A-P-E motif of subdomain II (aa 456-463) supporting the conclusion that SmTK6 is a CTK (supplementary data 1). Src kinase-characteristic sequence motifs also occur within the SH2 and SH3 domains. Within the N-terminal part of the SH3 domain of Src kinases the consensus sequence ALYDY is located, which concurs well with ALYSY in SmTK6 (aa 119 -123). At the C-terminus of Src SH3-domains occurs the motif PSNYV matching exactly to that of SmTK6 (aa 164 -168). Within the SH2 domains one histidine (His, H) (aa 233) and three arginine (Arg, R) residues occur at conserved positions (aa 186, 206, 234 in SmTK6; supplementary data 1), which are important for target protein interactions (31). However, SmTK6 also reveals features characteristic for Abl-kinases (6). Besides the absence of Tyr 416, a Tyr residue occurs at position 312 of SmTK6. This is one of two conserved Tyr residue typically found in the N-terminal part of Abl TK-domains (Fig. 1A, supplementary data 1). Comparing only the TK domain sequences of Abl and Src kinases by ClustalW analysis indicated that the overall structure of the catalytic domain of SmTK6 is more similar to Abl than to Src kinases (18). Abl kinases of Drosophila or Homo sapiens again possess an elongated C-terminus containing various combinations of characteristic binding regions for F-or G-actin, microtubules or DNA, all of which are absent from SmTK6 as well as a nuclear localization sequence, which was found in the Abl1 kinase of humans (Fig. 1A). Thus, SmTK6 is a Src-biased structural hybrid of both kinase classes representing a potential ancestor of Abl kinases.
To find additional support for the hybrid character a phylogenetic analysis was performed using sequence cassettes representing the regulatory regions SH3, SH2, and TK each for comparison. SmTK5, a Fyn kinase-like CTK of S. mansoni (32), was taken as outgroup. This analysis confirmed the Src/Abl intermediate position of SmTK6 supporting its ancestral role as a potential Abl precursor (Fig. 1B).
Inhibitor studies in Xenopus oocytes reveal an intermediate Src/Abl kinase activity of SmTK6-To elucidate whether SmTK6 possesses Src or Abl kinase-like activity, biochemical studies were performed in Xenopus oocytes. As shown before, schistosome kinases can be efficiently expressed in this system, and kinase activities proven by their capacity to induce GVBD as a resumption of meiosis (13,21,22,25,33). To be enzymatically active, kinases have to adopt an open conformation, which is usually acquired following interaction with an activated partner molecule such as a receptor. In the absence of an activating partner in Xenopus oocytes, the activity of a schistosome CTK expressed in this system can be achieved if its catalytic TK domain is expressed devoid of regulatory domains responsible for inactive conformation (13). Therefore, cRNA encoding the TK domain of SmTK6 (SmTK6-TK) was injected into Xenopus oocytes. Results showed that the expression of SmTK6-TK protein induced 100% GVBD in oocytes demonstrating the kinase activity of SmTK6-TK (Table 1A). This activity was completely abolished when the Src kinase-specific inhibitor Herbimycin A was added at a concentration of 10 µM. Herbimycin A was shown before to be able to block the activity of the Src kinase SmTK3, and to reduce male-induced mitotic activity in paired females as well as egg production in worms cultured in vitro (16,34). In the Xenopus oocyte assays, we confirmed that Herbimycin A was a potent inhibitor of SmTK3 that completely blocked its activity already at a concentration of 0.01 µM (Table 1A). In contrast, the kinase activity of SmAbl1-TK, the TK domain of an Abl-kinase homolog from S. mansoni (18) was insensitive to high doses of Herbimycin A (100% GVBD at 0.1 µM), and was only partially inhibited at concentrations of 1 µM (80% GVBD) and 10 µM (60% GVBD).
In an inhibitor swap-like experiment we also compared the effect of the Abl-inhibitor Imatinib on the capacity of SmTK6-TK and SmAbl1-TK to induce GVBD (Table 1B). This specific inhibitor has been successfully used in human cancer therapy (Gleevec, Novartis) to treat chronic myelogenous leukemia, which is caused by deregulation of the cAbl-kinase (35,36). Furthermore, in vitro culture studies with adult schistosomes demonstrated recently that Imatinib significantly affected schistosome morphology and physiology. Besides negative effects on gonad development and pairing stability, this inhibitor caused pathological alterations of the gastrodermis leading to parasite death (18). Whereas in Xenopus oocytes SmAbl1-TK activity was completely blocked at a concentration of 1 µM, the total inhibition of SmTK6-TK activity required 100 µM Imatinib, a concentration at which SmTK3-TK activity was only slightly reduced (by 10%; Table 1B).
As expected, these results confirmed the biochemical nature of SmTK3 as a Src-like kinase, but also the Abl kinase-like nature of SmAbl1. More importantly, these data demonstrated an inhibition profile of SmTK6 being intermediate to that of Src-and Abl kinases.
To find evidence for the role of SmTK6 in signaling processes, a search for potential interaction partners was performed.
Transmembrane receptors, a Src kinase, and a tumor suppressor molecule are potential upstream binding partners of SmTK6-Signaling molecules of the Src and Abl-kinase families each possess one SH2 domain, which is known to interact with upstream binding partners in signaling cascades. Using a bait construct expressing the SH2 domain of SmTK6, the S. mansoni adult stage Y2H cDNA-library (20) was screened. Expression of the bait was confirmed at the transcriptional level by RT-PCR analyses using RNA extracts from transformed yeast cells. Screening resulted in the identification of 77 initial prey clones, which underwent growth selection and β-gal filter assays (color selection) reducing the number to 29 clones. The isolation of the prey plasmids succeeded for 19 of these. Their inserts represented partial sequences of protein coding genes for which full-length (fl) cDNA sequences were detected in the genome data set of the S. mansoni sequencing project (37; www.sanger. ac.uk), or in the NCBI database (www.ncbi.nlm. nih.gov) using the obtained sequences for BlastX analyses. The results showed that four clone groups ( Table 2; A -D) were represented consisting of a transmembrane mucin homology group (named SmTmMuc1, A), the Src kinase SmTK3 (B), a tumor suppressor protein with homology to Discs-large (SmDLG, C), and proteins with no significant homology (D).
To confirm interactions of potential binding partners with known identity (SmTK3, SmDLG, and SmTmMuc1) and to determine their relative binding strengths in a comparative approach, yeast cells (AH109) were transformed with appropriate prey plasmids together with the bait construct SmTK6-SH2 pBridge. After transformation, all yeast clones survived growth and color selection. To quantify the relative strengths of interaction β-gal liquid assays were performed. The results again confirmed the observed interactions with SmTK6 and demonstrated the strongest affinity between the SmTK6 SH2domain and SmTK3. The interactions between the SmTK6 SH2-domain and SmDLG or SmTmMuc1 were considerably weaker ( Fig. 2A).
Literature data have indicated that Src kinases can act in concert with Syk kinase to participate in membrane receptor complexes, which among others contain receptor tyrosine kinases (RTKs) (38). SmVKR1 of S. mansoni is a RTK and was shown to be expressed in the ovary of females (23), thus co-localizing with SmTK6 and SmTK4 (13,17). Therefore, we investigated whether SmVKR1 may interact with these CTKs. To this end direct binding studies were performed in the Y2H system with the intracellular part of SmVKR1 and the protein interaction domains of SmTK6, SmTK3 or SmTK4. Yeast cells (AH109) were transformed with the prey construct SmVKR1 C-term pACT2 and with bait plasmids containing both or individual SH3 and SH2 domains of SmTK6 or SmTK3, or the tandem SH2 domain of SmTK4. Following transformation, all yeast clones survived growth and color selections indicating that all three kinases were able to bind by their SH2/SH3 domains to the intracellular part of SmVKR1. This was confirmed by β-gal liquid assays, which also provided a first hint to the relative strengths of the observed interactions demonstrating the strongest interaction of SmTK6 with the C-terminus of SmVKR1 (Fig. 2B). As expected, the interaction of the SH2-domain of SmTK6 was stronger compared to the interaction of its SH3 domain or the combined SH3SH2 domains. In contrast, the combined SH3SH2 domains of SmTK3 interacted stronger with the intracellular part of SmVKR1 than the individual SH2 or SH3 domains of SmTK3. Finally, the interaction of the tandem SH2-domain of SmTK4 to SmVKR1 was stronger than the interaction of SmTK3-SH3SH2, but weaker than the interaction of SmTK6-SH2 with SmVKR1.
Co-immunoprecipitation experiments confirm SmTK6-SmTK3 and SmTK6-SmVKR1 interactions-Previous studies showed that SmTK3 and SmVKR1 are expressed in the gonads of schistosomes co-localizing in the ovary (15,23). Since SmTK4 and SmTK6 are also transcribed in the ovary of females, and since by Y2H library screening SmTK6 was found as a binding partner, whose interaction with SmTK4 was confirmed by co-immunoprecipitation experiments (13), a kinase complex acting in the gonads was first hypothesized (13,14).
To provide further evidence for such a complex, co-immunoprecipitation experiments were performed with the strongest interaction partners SmTK3 and SmVKR1. To this end the Xenopus oocyte expression system was used again.
For interaction analyses of SmTK6 and SmVKR1, Flag/V5-tagged SmTK6-fl and the active kinase of SmVKR1 (myc-tagged SmVKR1 YYRE -C-term) constructs were coexpressed in oocytes. SmVKR1 was detected in anti-myc immunoprecipitates when injected alone or with SmTK6 following anti-myc Western blotting (Fig. 3B, a, lanes 2 and 3). Similarly, SmTK6 was detected in anti-V5 immunoprecipitates when injected without or with SmVKR1 (Fig. 3B, d, lanes 1 and 3) confirming the correct expression of both proteins in oocytes. Results of the analyses of oocyte extracts containing both SmVKR1 and TK6 showed that V5-tagged SmTK6 was contained in anti-myc precipitates (Fig. 3B, b,  lane 3) and, inversely, myc-tagged SmVKR1 was present in anti-V5 precipitates (Fig. 3B, c, lane 3) demonstrating the interaction between the two molecules. In summary, these coimmunoprecipitation experiments confirmed the physical interactions of SmTK6 with both SmTK3 and SmVKR1.
SmTK6 is activated by SmVKR1 or SmTK3 and induces GVBD in Xenopus oocytes-To confirm that SmVKR1 not only interacts with, but also activates SmTK6, cRNAs encoding the full-length version of SmTK6 (SmTK6-fl) or its SH2-SH3 protein interaction domains (SmTK6-SH3SH2) were co-injected into Xenopus oocytes with SmVKR1 YYRE , the constitutively active kinase mutant of SmVKR1. Comparative GVBD assays were performed ( Table 3). As expected, the expression of SmTK6-fl alone did not lead to GVBD due to its inactive kinase conformation. SmTK6-SH2SH3 alone was also unable to induce GVBD, in contrast to the SmTK6-TK catalytic domain, which induced 100% GVBD ( Table 1). The expression of SmVKR1 YYRE alone also led to GVBD, whereas wild-type SmVKR1 in the absence of ligand had no effect as previously shown (25). The results showed that the co-expression of SmTK6-fl with SmVKR1 YYRE did not affect GVBD induced by the SmVKR1 active kinase. However, when the SH2SH3 domains of SmTK6 were co-expressed with SmVKR1 YYRE , GVBD was totally inhibited, suggesting that binding of protein interaction domains of SmTK6 to SmVKR1 could specifically compete with partners found by SmVKR1 in oocytes preventing its GVBDinductive potential. Also, the demonstration that a "kinase-dead" variant of SmTK6-fl (SmTK6fl-ko) was able to block the SmVKR1 YYREinduced GVBD in oocytes confirmed that molecular interactions effectively occur between SmTK6 and SmVKR1 leading possibly to SmTK6 kinase activation, as a step of the signaling cascade required for oocyte maturation.
Since SmTK3 represented the other potent interaction partner of SmTK6, we investigated its potential to alternatively induce SmTK6 catalytic activity, too. SmTK6-fl or SmTK3-fl alone were not able to induce GVBD in Xenopus oocytes (Table 4), while their co-expression elicited GVBD. However, the co-expression of SmTK3-fl with the kinase-dead variant SmTK6fl-ko or with the SmTK6-SH3SH2 domains had no inductive effects on oocyte maturation, indicating that the full-length version of SmTK6 is required for activation. In contrast, it appeared that the presence of a full-length and enzymatically active version of SmTK3 was not required to obtain GVBD in this system since the co-expression of a kinase-dead variant of SmTK3 (SmTK3-fl-ko) or of only the TK domain of SmTK3 with SmTK6-fl induced oocyte maturation (Table 4). These results indicated that SmTK6 gets activated independently of the kinase activity of SmTK3. Indeed, the ability of the dead-kinase SmTK3-flko to activate SmTK6-fl was corroborated by the observation that Herbimycin A (used at a concentration already shown to block SmTK3 kinase activity completely; Table 1), did not inhibit GVBD induced by the co-expression of SmTK3-TK with SmTK6-fl. Finally, these results provided evidence that the kinase potential of SmTK6 leading to GVBD is positively controlled by its interaction with SmTK3 (Table 4).
Transcriptional analyses demonstrate similar activity profiles of the interaction partners-RT-PCR analyses showed that SmTK6 transcription observed in males and females corresponded to the transcriptional profile of SmDLG, however, differences were observed for the larval stages since SmDLG transcripts were detected only in miracidia, but not in cercariae (supplementary data 2). In contrast, SmTmMuc1 was found to be transcribed in the adult stages as well as in both larval stages (Hahnel et al. in preparation), which corresponded also to the transcriptional profiles of SmTK3 (15) and SmTK4 (17). Co-localization of SmTK6 with SmTK4 transcripts in the testes and the ovary of adult schistosomes had already been demonstrated before (13). As shown by in situ hybridization, SmDLG co-localized with SmTK6 in the gonad tissues of adult worms ( Fig. 4 A-C). This applies also to SmTmMuc1 transcripts whose occurrence was observed in the gonads, but also in other tissues (Hahnel et al., in preparation). Especially the transcription pattern in the reproductive organs of SmDLG and SmTmMuc1 corresponded to that of SmTK6, and there is additional co-localization with SmTK4 in the ovary and in the testes. Finally, transcripts of SmVKR1 used as positive control were detected as expected exclusively in the ovary of females (Fig. 4 D, E), which corresponded to previous data (22). Sections of control worms hybridized with sense transcripts did not show any signal. The localization results indicated that the ovary is the organ where SmTK6 and SmVKR1 co-localize with all other molecules investigated in this study, but also with SmTK3 and SmTK4.

DISCUSSION
Structural and phylogenetic analyses performed in the present study provided conclusive evidence that SmTK6 represents an exceptional hybrid kinase taking an intermediate evolutionary position between Src and Abl kinases being the first hybrid kinase detected in a helminth. Members of both kinase families have been found in the majority of metazoans, which suggests that their structure and function were fixed early in evolution. Vertebrate genomes encode two closely related paralogs, Abl1 and Abl2, which may have originated from a gene duplication event. Invertebrate metazoans such as Caenorhabditis elegans or Drosophila possess a single Abl gene, which shows strong conservation through the SH3-SH2-TK cassette and an elongated C-terminus with an actinbinding domain (6). The choanoflagellate Monosiga brevicollis, a unicellular protist, encodes two Abl kinases with a shortened Cterminal end without actin-binding domain. This suggested an early origin for Abl kinases, with the addition of extended carboxy termini during the metazoan radiation (6). In conformity with this hypothesis schistosomes, and may be other trematodes, seem to be gap-filling in evolutionary terms by possessing two Abl genes with elongated C-termini. But none of these contains a conserved actin-binding domain (Fig. 1A). SmTK6 has no actin-binding domain as well and exhibits the shortest C-terminus found so far for Src or Abl kinases. Within its C-terminus, the Src characteristic Tyr 527 occurs, but the regulatory Tyr 416 is missing. Instead, one of two Abl kinase-specific Tyr residues occur (Fig. 1A). This finding and the results of the phylogenetic analyses suggested a hybrid character of this unusual kinase. Functional evidence in support of this hypothesis was obtained by GVBD assays in Xenopus oocytes expressing the catalytic TK domains of SmTK6 and other schistosome kinases representing members of the Src (SmTK3) or Abl (SmAbl1) kinase families (15,18). Using inhibitors specific for each of these CTK families demonstrated the hybrid character also at the biochemical level. SmTK6-TK-induced GVBD was inhibited by the Src-kinase inhibitor Herbimycin A at a 1000x higher concentration than that needed to inhibit SmTK3-TK-induced GVBD. The Abl kinase inhibitor Imatinib was also able to completely block SmTK6-TK activity, but at a 100x higher concentration than that needed to inhibit SmAbl1-TK-induced GVBD. Thus, the inhibition profile of SmTK6 was found to be intermediate between Src and Abl kinases. In a previous study it was shown that 21 aa residues of the human Abl kinase interact with Imatinib (39). Of these 18 are conserved in SmAbl1 according to our analysis, but only 15 in SmTK6 (18). This difference possibly explains the reduced inhibitory effect of Imatinib on SmTK6 compared to SmAbl1. Among the differences is an aa substitution next to the conserved DFG motif within the catalytic TK domain. In contrast to Abl kinases of Drosophila, C. elegans or H. sapiens, which exhibit a conserved ADFGL (aa 381-385) sequence motif, SmTK6 has SDFGL instead (aa 435-438 in SmTK6; supplementary data 1). Since high-affinity binding of Imatinib requires a flipped DFG motif (40), the non-synonymous A/S substitution may contribute to this conformational change and negatively influence inhibitor-binding.
As results from Y2H library screening schistosome homologs of the known Src kinase SmTK3 (15), the tumor suppressor protein DLG, and a novel transmembrane mucin were found as potential interaction partners of SmTK6. RT-PCR analyses showed overlapping transcriptional profiles of all genes in adult stages, and in situ hybridizations finally demonstrated co-localization in the gonads of adults. Interactions of SmTK6 were also found with the colocalizing SmVKR1 (23). This RTK may not have been represented in, or picked-up from the library due to a potential competition between membrane localization signals within its sequence and the NLS-sequences of the prey vector destined to ensure nuclear interaction and reporter gene induction.
DLG is a tumor suppressor protein and a prototype of a growing family of proteins collectively termed membrane-associated guanylate kinase homologs (MAGUKs). Genetic studies in Drosophila revealed that three tumor suppressors, DLG, Scribble, and Lethal giant larvae (LGL) co-localized to the basolateral region of epithelial cells cooperatively regulating cell polarity, junction formation and cell growth during oogenesis (41). Studies in vertebrates and C. elegans demonstrated the evolutionary conservation of some of their functions (42), and new evidence has indicated a role of MAGUKs in asymmetric cell division (43), a characteristic feature of germ-cells (44). A presumptive interaction of Src-kinases and PDZ (Post synaptic density SD95/SAP90, DLG, Zonula occludentes1) domain-containing proteins such as DLG was shown recently (45). Since the partial SmDLG clone contained a PDZ domain, interaction with SmTK6 may have been mediated by this domain. Recently we found a S. mansoni LGL homolog (SmLGL) whose transcripts were also localized in the reproductive organs (Buro et al., in preparation). Since two schistosome homologs of Scribble exist in the genome (Smp_180220.2; Smp_104030.2), all members of a co-operatively acting molecular network are present in this parasite that have been shown before in Drosophila to play roles during oogenesis (41).
As a group of high molecular weight glycoproteins, mucins are divided into secreted and membrane-bound forms containing a single transmembrane domain (46). Members of the latter class are involved in cellular signaling events, and they contribute to carcinogenesis (47). Among others, transmembrane mucins such as human MUC1 have the potential to interact with RTKs such as EGFRs (48). The colocalization of SmTmMuc1 with SmTK6 and other schistosome kinases including SmVKR1 suggests a role in signal transduction processes in the gonads of schistosomes in concert with further signaling molecules (Hahnel et al., in preparation).
Src kinases are known to be involved in signaling pathways regulating cell proliferation and differentiation (1)(2)(3). Often they are parts of large complexes consisting of different cellular, membrane-associated, and transmembrane signaling proteins. SmVKR1 (previously called SmRTK-1) was described as an unusual schistosome RTK composed of an extracellular Venus Flytrap Module (VFT; a ligand-binding domain in class 3 GPCRs) linked by a single transmembrane domain to a TK domain, which displayed similarity to that of insulin receptors (23). VKRs have been found in different invertebrates, particularly in insects (24). As in schistosomes, VKRs are mainly expressed in female gonads, indicating a putative function of these receptors in reproduction and/ or development. It has been hypothesized that SmVKR1 might be involved in the recognition of a male pheromone signal necessary for the development and maturation of the ovary since it possesses a VFT module, also present in mammalian pheromone receptors (14,23,49). SmVKR1 transcripts were predominantly found in mature oocytes of female schistosomes by in situ hybridization, which coincided with the transcript localizations of SmTK4 (17), SmTK3 (15), and SmTK6 (13). In addition to the SmTK4-SmTK6 binding confirmed previously (13), we showed in this study by co-immunoprecipitation experiments that SmTK6 interacts with SmTK3 as well as with SmVKR1. In addition, GVBD assays in Xenopus oocytes demonstrated that SmTK6 interacted with and is likely activated by catalytically active SmVKR1. Furthermore, we showed that SmTK3 is also able to activate SmTK6. For activation, binding to SmTK3 alone was sufficient and independent from the catalytic potential of SmTK3. We assume that SmTK6 changes into an open conformation upon SmTK3 binding as a prerequisite to become active inducing GVBD in Xenopus oocytes. A question still to solve in further studies is whether SmTK6 gets phosphorylated and finally activated by SmVKR1, and whether SmTK3 in such a scenario plays a chaperone-like role supporting the conformational change of SmTK6 for further catalytic interactions within the postulated multikinase complex.
Another attractive goal of this study was to knock-down SmTK6 by RNA interference (RNAi) to study the phenotype among other by confocal laser scanning microscopy (CLSM). This approach has been successfully applied before to characterize schistosome kinases such as SmTK4 (13). Although standard protocols (dsRNA; 13, 50) as well as novel approaches using different sets of specifically designed siRNAs (Beckmann et al., in preparation) were applied by electroporation in adult schistosomes maintained in vitro, we obtained neither knockdown effects at the transcriptional level (determined by qPCR) nor phenotypic changes (as determined by CLSM; 13). SmTK6 may belong to the group of genes, which according to recent studies in this field were described as non-knockable genes (50).
With respect to their interactions and colocalizations in the reproductive organs SmTK6, SmVKR1, SmTK3, SmTK4, and SmDLG seem to be parts of a complex scenario (Fig. 5). In previous studies, evidence was obtained that SmTK3 interacts with SmTK4, and that both may be members of a CTK complex (14). Together with the Rho-GTPase SmRho1 and the diaphanous homolog SmDia, SmTK3 was suggested to be part of integrating RTK-and GPCR signaling pathways, which organize the actin cytoskeleton within the gonads of schistosomes (20). Homologs of a MAP kinaseactivating protein (PM20/21) and mapmodulin were found before as downstream partners of SmTK4, and SmTK6 as its strongest upstream partner (13). CTK members of this multi-kinase complex such as SmTK6 are probably controlled by SmVKR1 (Fig. 5). SmDLG as further binding partner of SmTK6 may become activated upon complex formation. Subsequently, SmDLG may interact with other MAGUKs such as SmLGL and Scribble to control processes of cell growth and/or cell polarity.
Elucidating cellular processes leading to gonad development in schistosomes along with the increasing knowledge of molecules involved will help to find novel strategies to fight this blood-fluke. To this end kinases represent interesting targets to identify inhibitors affecting developmental processes. Such inhibitors could already represent approved drugs (18), or they can serve at least as lead structures for drug design or vaccination (51). This is an urgent need in the light of the fact, that Praziquantel is the only widely used drug to treat schistosomiasis evoking the fear of emerging resistance (52,53).

FIGURE 4.
Non-conserved regions of SmDLG (A-C) and SmVKR1 (D, E) served as templates to synthesize digoxigenin (DIG)-labeled antisense transcripts used as probes for in situ hybridization. SmVKR1 signals were exclusively detected in the ovary (o), whereas SmDLG signals were detected in the ovary and the vitellarium (v) of the female as well as in the testes (t) of the male. For control, a DIG-labeled sense probe was used (F-H). [scale bar: 50 µm] FIGURE 5. In S. mansoni the CTKs SmTK3, SmTK4, and SmTK6 may be members of a trimeric complex, which interacts with the RTK SmVKR1. Results of a previous study had already indicated that SmTK3 also interacted with the diaphanous homolog SmDia, which is a binding partner of the Rho-GTPase SmRho1. Both SmDia and SmRho1 were suggested to be part of cooperative RTK and GPCR signaling pathways integrating at SmDia to organize the actin cytoskeleton within the gonads of schistosomes (20). As downstream partners of SmTK4 a MAP kinase-activating protein (PM20/21) and mapmodulin were found, which may be involved in cytoskeleton reorganization and mitosis (13). SmDLG as a binding partner of SmTK6 may become activated upon complex formation and may subsequently interact with SmLGL and Scribble to control processes of cell growth and/or cell polarity.

TABLES TABLE 1. Influence of Herbimycin A or
Imatinib on the capacity of the catalytic TK domains of SmTK3, SmTK6 or SmAbl1 expressed in Xenopus oocytes to induce GVBD. A. SmTK3-TKinduced GVBD (numbers represent % GVBD; mean of two independent experiments) was completely blocked by Herbimycin A at a concentration of 0.01 µM. Total inhibition of SmTK6-TK-induced GVBD required 10 µM. At this concentration SmAbl1-TK was still active (60%). B. Symbolism as in A. Imatinib completely blocked SmAbl1-TK at 1 µM. At this concentration, the activities of SmTK3-TK or SmTK6-TK were not affected. Whereas SmTK3-TK was still active (90%) at 100 µM Imatinib, SmTK6-TK was totally inhibited at this concentration. [-= not determined]