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

Svenja Beckmann,Steffen Hahnel,Katia Cailliau,Mathieu Vanderstraete,Edith Browaeys,Colette Dissous,Christoph G Grevelding

doi:10.1074/jbc.m110.210336

Abstract

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.

Highlights

SmTK6 was identified as interaction partner of SmTK4
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
Compared with other Src kinases, SmTK6 possesses only one of the typical Tyr residues with regulatory function (Tyr-562). It is positioned within the C terminus close to the tyrosine kinase (TK) domain at a conserved position compared with Tyr-527 of human c-Src (Fig. 1A and supplemental data 1)

Summary

Background

SmTK6 was identified as interaction partner of SmTK4. Results: SmTK6 is a Src/Abl hybrid kinase and interacts with the uncommon SmVKR1 and SmTK3. In Xenopus oocytes, it was shown that SmVKR1 and SmTK3 were able to activate SmTK6 enzymatic activity indicating its functions in a receptor tyrosine kinase signal transduction cascade These results demonstrate an intermediate but Srcbiased profile of the unusual kinase SmTK6. We identified upstream-binding partners 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 co-immunoprecipitation experiments, which confirmed SmTK6-SmTK3 as well as SmTK6-SmVKR1 interactions, germinal vesicle breakdown (GVBD) assays in Xenopus oocytes demonstrated that SmTK6 can be activated by SmVKR1 or SmTK3 These results reinforce previous suggestions of a multikinase 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

RESULTS

DISCUSSION