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Fig 1. SmShb belongs to the Shb protein family.(A) SmShb is a protein of 1159 amino acids with a Proline-rich domain (AA288-407, in green) in its N-terminal part and an SH2 domain in its C-terminal end (AA1054-1139, in blue). (B) A phylogenetic tree was generated using the maximum likelihood method under the JTT+G+I model with a bootstrap test (1000 replicates). The tree was generated using the alignment of proteins from Shb (Mus musculus MmShb NP_001028478.1, MmShf NP_001013851.2, MmShe AAI09363.2, MmShd BAA33805.1; Homo sapiens HsShb CAA53091.1, HsShf Q7M4L6.2, HsShe Q5VZ18.1, HsShd Q96IW2.1; Camponotus floridanus CfShf EFN71155.1; Anopheles gambiae AgShb XP_309808.4; Clonorchis sinensis CsShf GAA56149.1, CsShb GAA50136.1; Schistosoma mansoni SmShb AFH41560.1; Echinococcus granulosus EgShf CDS15334.1 and E. multilocularis SmShf CDJ02881.1), Grb2 (S. haematobium ShGrb2 KGB37632.1; SmGrb2 CCD60977.1; S. japonicum SjGrb2 CAX75443.1; CsGrb2 GAA48411.1; EgGrb2 CDS18126.1; EmGrb2 CDJ01173.1; Drosophila melanogaster DmGrb2 NP_476858.1; HsGrb2 CAG46740.1 and MmGrb2 AAB40022.1), Grb7 (HsGrb7 BAA29059.1, HsGrb10 NP_005302.3, HsGrb14 AAC15861.1; MmGrb7 NP_034476.1, MmGrb10 AAB53687.1, MmGrb14 AAF43996.1), SHC (SmSHC CCD76305.1; ShSHC3 KGB39408.1; EmSHC3 CDJ05613.1; EgSHC3 EUB61901.1; DmSHC NP_524683.2; Xenopus laevis XlSHC1 NP_001083932.1; HsSHC1 NP_892113.4; MmSHC1 NP_001106802.1), and Nck (SmNck2 CCD77399.1; ShNck1 KGB32228.1; EmNck CDJ02918.1; EgNck CDS15372.1; HsNck2 NP_001004720.1, HsNck1 NP_001278928.1; MmNck2 NP_035009.3, MmNck1 NP_035008.2; XlGrb4 NP_001083313.1, XlNck AAH80058.1) families.
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Fig 2. SmShb interacts specifically with activated SmVKR1.(A) Direct interaction in S. cerevisiae yeasts: AH109 yeasts expressing Gal4AD-fused SmShb were mated with Y187 yeasts expressing only Gal4DBD (pGBKT7), Gal4DBD-fused ICD YYxE (intracellular domain with active kinase) of SmVKR1, SmVKR2, SmIR1 or SmIR2, or Gal4DBD-fused ICD DK (dead kinase) of SmVKR1. Diploids were allowed to grow on a minimal SD -Leu/-Trp medium and diploids expressing interacting proteins were then selected on SD -Leu/-Trp/-His/-Ade medium. Only yeasts expressing SmShb and SmVKR1 ICD YYRE grew on the selective medium. (B) cRNAs encoding Myc-tagged SmVKR1 ICD WT or YYRE were co-injected in Xenopus oocytes with cRNA encoding HA-tagged SmShb. Oocytes were incubated for 5h in ND96 medium and lysed. Proteins from soluble extracts were immunoprecipitated (IP) by anti-Myc or anti-HA antibodies and immune complexes were analyzed by Western Blot (WB) to detect SmShb (130 kDa) and SmVKR1 ICD (68 kDa) with anti-HA or anti-Myc antibodies.
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Fig 3. SmVKR1 interacts with SH2 domain and phosphorylates SmShb.cRNAs encoding Myc- SmVKR1 ICD YYRE or SmVKR2 ICD YYRE were injected in Xenopus oocytes with HA- SmShb, SmShbÎSH2 (lacking the SH2 N-terminal domain) or the isolated domain SH2SmShb. Oocytes were incubated for 5h in ND96 medium and lysed. Proteins from soluble extracts were immunoprecipitated (IP) by anti-HA antibodies and analyzed by Western Blot (WB) with anti- HA and anti-Myc antibodies. HA-SmShb (130 kDa), HA-SmShbÎSH2 (118 kDa) and HA-SH2SmShb (10 kDa) were detected by anti-HA and Myc- SmVKR1 ICD YYRE (68 kDa) was detected in complexes made with HA-SmShb and HA-SH2SmShb but not with HA-SmShbÎSH2. Proteins from soluble extracts were also immunoprecipitated by anti-Myc antibodies and analyzed by Western Blot (WB) with anti- Myc, anti-HA and anti-phosphotyrosine (PY20) antibodies. Myc- SmVKR1 ICD YYRE (68 kDa) and SmVKR2 ICD YYRE (81 kDa) were detected and both HA-SmShb and HA-SH2SmShb were detected in complexes made with Myc- SmVKR1 ICD YYRE. Anti-phosphotyrosine antibodies (PY20) confirmed the phosphorylation of SmVKR1 ICD YYRE and SmVKR2 ICD YYRE and revealed that of SmShb (130kDa) in the immune complexes formed with SmVKR1 ICD YYRE.
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Fig 4. The juxtamembrane tyrosine (Y979) of SmVKR1 is required for SmShb binding.cRNAs encoding the intracellular domains (ICD) of Myc- SmVKR1 YYRE or SmVKR1 YYRE Y979F were co-injected in Xenopus oocytes with HA-SmShb and soluble extracts were immunoprecipitated (IP) by anti-Myc or anti-HA antibodies. Immune complexes were analyzed by Western Blot (WB) using anti-Myc or anti-HA antibodies. SmShb co-precipitated with SmVKR1 ICD YYRE but not with SmVKR1 ICD YYRE Y979F.
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Fig 5. SmShb interacts with ligand-activated SmVKR1 and SmVKR2 receptors.(A) cRNA encoding V5-tagged SmVKR1 WT or SmVKR1 Y979F were injected with or without cRNA encoding HA-tagged SmShb in Xenopus oocytes. Following their expression, full-length receptors were activated by L-Arg to induce their auto-phosphorylation [11]. Proteins were then analysed by immunoprecipitation (IP) using anti-V5 or anti-HA antibodies followed by western blot (WB) using anti-V5, anti-HA or PY20 (anti-tyrosine phosphorylation) antibodies. Results demonstrated that only phosphorylated SmVKR1 WT bound to and phosphorylated SmShb as revealed by PY20 antibodies. (B) cRNA encoding V5-tagged SmVKR2 WT or SmVKR2 F949Y were injected with or without cRNA encoding HA-tagged SmShb in Xenopus oocytes. Following their expression, full-length receptors were activated by Ca++ to induce their auto-phosphorylation [11]. Proteins were then analysed by immunoprecipiration (IP) using anti-V5 or anti-HA antibodies followed by western blot (WB) analysis using anti-V5, anti-HA or PY20 (anti-tyrosine phosphorylation) antibodies. Results indicated that phosphorylated SmVKR2 WT did not bind SmShb whereas the mutated V5-SmVKR2 F949Y bound to and phosphorylated SmShb.
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Fig 6. SmVKR-SmShb interaction induces JNK pathway.Xenopus oocytes were co-injected with cRNAs encoding V5-SmVKR1 WT or Y979F or V5-SmVKR2 WT or F949Y and SmShb and incubated with 1μM L-Arginine or 1mM Ca++ respectively. Oocyte lysates were analyzed by Western blot to detect phosphorylation of ERK2, Akt, S6K and JNK as described in Materials and Methods. In the absence of SmShb, SmVKR1 WT and Y979F activated ERK2, Akt, S6K and JNK pathways whereas SmVKR2 WT and F949Y activated ERK2, Akt, S6K but not JNK. However, in the presence of SmShb, SmVKR1 WT and SmVKR2 F949Y did no more activate ERK2, Akt, S6K. While JNK pathway was maintained with SmVKR1 WT, it was specifically induced by SmShb in the case of the SmVKR2 F949Y mutated and able to bind SmShb. In both cases, GVBD dependent on ERK2 and Akt activation was inhibited. SmShb did not affect signaling by SmVKR1 Y979F or SmVKR2 WT, which were not able to interact with SmShb.
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Fig 7. SmShb is a partner of the JNK pathway.cRNA (40 ng) of SmVKR1(A) or SmVKR2 F949Y (B) were injected in oocytes without or with cRNA (25 ng) encoding SH2SmShb or SH2-deleted SmShb (SmShbÎSH2). After two hours, a second injection of cRNA encoding SmShb (35 ng) was performed. Oocytes were further incubated for 3 hours then lysed. JNK phosphorylation was detected in oocyte lysates by western blotting using anti-phospho JNK antibodies. Pre-incubation of the SH2 domain with SmVKR1 and with SmVKR2 F949Y blocked the capacity of SmShb to induce JNK pathway.
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Fig 8. Localization of SmShb transcripts in sections of paired adult worms by in situ hybridization.SmShb transcripts were detected in mature oocytes (A), in testes (B) and lightly in the vitellarium (C). Sense probe of SmShb was used as control in D, E and F. (m: male; f: female; mo: mature oocytes; t: testes; v: vitellarium; scale bar = 20μm)
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Fig 9. Effects of SmShb knock-down by RNA interference in adult worms.Worm couples were electroporated and incubated for 7 days either with dsSmShb or irrelevant dsLuc (control) as described in Materials and Methods. (A) Levels of SmShb transcripts were determined by quantitative RT-PCR in each worm sample. Interference led to a reduction of about 80% in the level of SmShb transcripts compared to controls. Statistical analysis was performed using the Studentâs t-test and values are expressed as mean ± SEM of four determinations (*** p<0.001). (B) CLSM images of whole-mount preparations of male (a, b) or female (c, d) worms issued from worm couples electroporated with dsLuc (a, c) or dsSmShb (b, d). Accumulation of sperm in testicular lobes is observed in dsSmShb-treated males (b). Less mature oocytes were present in the posterior part of the female ovary (d). (ov: ovary; mo: mature oocytes; io: immature oocytes; t: testes; sv: seminal vesicle; arrows indicate mature sperm; scale bar = 20μm)
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