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The signal transduction protein SmTK4 from Schistosoma mansoni belongs to the family of Syk kinases. In vertebrates, Syk kinases are known to play specialized roles in signaling pathways in cells of the hematopoietic system. Although Syk kinases were identified in some invertebrates, their role in this group of animals has not yet been elucidated. Since SmTK4 is the first Syk kinase from a parasitic helminth, shown to be predominantly expressed in the testes and ovary of adult worms, we investigated its function. To unravel signaling cascades in which SmTK4 is involved, yeast two-/three-hybrid library screenings were performed with either the tandem SH2-domain, or with the linker region including the tyrosine kinase domain of SmTK4. Besides the Src kinase SmTK3 we identified a new Src kinase (SmTK6) acting upstream of SmTK4 and a MAPK-activating protein, as well as mapmodulin acting downstream. Their identities and colocalization studies pointed to a role of SmTK4 in a signaling cascade regulating the proliferation and/or differentiation of cells in the gonads of schistosomes. To confirm this decisive role we performed biochemical and molecular approaches to knock down SmTK4 combined with a novel protocol for confocal laser scanning microscopy for morphological analyses. Using the Syk kinase-specific inhibitor Piceatannol or by RNAi treatment of adult schistosomes in vitro, corresponding phenotypes were detected in the testes and ovary. In the Xenopus oocyte system it was finally confirmed that Piceatannol suppressed the activity of the catalytic kinase domain of SmTK4. Our findings demonstrate a pivotal role of SmTK4 in gametogenesis, a new function for Syk kinases in eukaryotes.
Figure 1. Domain structure of SmTK4.SmTK4 contains an N-terminal tandem SH2-domain (aa 48–143 and aa 201–292; A: interdomain) followed by a linker region (linker) and a C-terminally located, catalytic tyrosine kinase domain (TK, aa 881–1147). Black bars indicate the relative position of the bait constructs SmTK4-SH2SH2 (upstream screening) and SmTK4-linker+TK (downstream screening).
Figure 2. Comparative β-Gal liquid assays to determine the relative binding strengths of SmTK4 upstream interaction partners.For this analysis, yeast cells (strain AH109) were re-transformed with one representative prey clone of each group together with the bait SmTK4-SH2SH2 pBridge (light grey) or SmTK4-SH2SH2 + SmTK3-TK pBridge (dark grey). The tested clones were (from left to right): SmTK6 (Src kinase), SmTK3 (Src kinase), a homolog of a dipeptidyl peptidase III, and a homolog of a nonsense mRNA reducing factor (NORF1). As control, untransformed yeast cells were used (control). The statistical evaluation of three independent measurements of β-Gal activity (n = 3) is shown (error bars are indicated). Student's t-test (two-tailed): *p<0.002.
Figure 3. Co-immunoprecipitation of SmTK4 and SmTK6.The tandem SH2-domain of SmTK4 was expressed with a FLAG-tag, and the sequence of SmTK6 with a cMyc-tag in yeast. Co-IPs were done with lysates from yeast (strain YPH501) expressing both proteins using anti-FLAG- (F) or anti-cMyc antibodies (M). After co-IP, the proteins were separated on a SDS-PAGE and blotted onto a nitrocellulose membrane. A: INDIA-Ink staining of the nitrocellulose membrane (N: negative control without antibody, L: yeast lysate before immunoprecipitation). B: Detection of SmTK6 full-length-cMyc (55 kDa) and SmTK4-SH2SH2-FLAG (31 kDa) by Western blots using anti-cMyc or anti-FLAG-tag antibodies, respectively.
Figure 4. In situ-hybridization experiments localizing SmTK6, MAPK activating protein, and mapmodulin transcripts.Representative sections (5 µm) of adult schistosome couples (males and females are indicated), which were hybridized with DIG-labeled antisense-RNA probes of SmTK6 (A–C), MAPK-activating protein (D–F), or mapmodulin (G–I). For control, a DIG-labeled sense-RNA probe of mapmodulin was used (J–L). mRNA transcripts of all genes were detected in the ovary (o), the testes (t), and the parenchyma (p). No MAPK-activating protein gene-transcripts or mapmodulin transcripts were found in the vitellarium (v), but weak SmTK6 signals were observed in this organ. In the muscles (m), the subtegument (s), or the gastrodermis (ga), no signals were detected. [g, gut; gy, gynaecophoric canal; scale bar: 50 µm.]
Figure 5. Comparative β-Gal liquid assays to determine the relative binding strengths of SmTK4 downstream interaction partners.Yeast cells (strain AH109) were re-transformed with one representative prey clone of the groups A–D together with the bait SmTK4-linker+TK + SmTK3-TK pBridge (white), SmTK4-linker+TK pBridge (light grey), SmTK4-linker pBridge (dark grey), or SmTK4-TK pBridge (black). The tested clones were (from left to right): a homolog of a small HSP 25, a caspase 3 homolog, a putative MAPK-activating protein (PM20/PM21), and a mapmodulin homolog. The statistical evaluation of six independent measurements of β-Gal activity (n = 6) is shown (error bars are indicated). Student's t-test (two-tailed): *p<0.0001, **p<0.005, *** p<0.05.
Figure 6. Morphology of the testes and ovary of Piceatannol- or dsRNA-treated S. mansoni.Confocal scanning laser microscope images of carmine red-stained whole-mount preparations of S. mansoni couples (A, C, E, G: males; B, D, F, H: females) treated with Piceatannol (C, D) or dsRNA (G, H). A, B: control worms treated with DMSO only; C, D: worms treated for 6 days with 70 µM Piceatannol; E, F: control worms after electroporation without dsRNA; G, H: worms after electroporation with SmTK4 dsRNA. [e: egg, io: immature oocytes, mo: mature oocytes, od: oviduct, vd: vitelloduct; asterisk: sperm vesicle, arrows: mature sperms; scale bar: 40 µm]
Figure 7. Influence of Piceatannol-treatment on egg production of S. mansoni couples.For this, 20 worm couples each were cultured for 7 days in the absence (DMSO control) or presence of 70 µM Piceatannol, and the egg numbers were determined at a daily basis. The statistical evaluation of four independent experiments (n = 4) is shown (error bars indicated). Student's t-test (two-tailed): *p<0.01.
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