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The formin-homology protein SmDia interacts with the Src kinase SmTK and the GTPase SmRho1 in the gonads of Schistosoma mansoni.

Quack T, Knobloch J, Beckmann S, Vicogne J, Dissous C, Grevelding CG - PLoS ONE (2009)

Bottom Line: The Src-like tyrosine-kinase SmTK3 of Schistosoma mansoni is expressed in the gonads, and its pharmacological inhibition reduces mitogenic activity and egg production in paired females in vitro.Among the binding partners found was a diaphanous homolog (SmDia), which was characterized further.Furthermore, the FH1 and inter domain region of SmDia have been discovered as binding sites for the SH3 and unique site domains of SmTK3, respectively.

View Article: PubMed Central - PubMed

Affiliation: Institute for Parasitology, Justus-Liebig-University, Giessen, Germany.

ABSTRACT

Background: Schistosomiasis (bilharzia) is a parasitic disease of worldwide significance affecting human and animals. As schistosome eggs are responsible for pathogenesis, the understanding of processes controlling gonad development might open new perspectives for intervention. The Src-like tyrosine-kinase SmTK3 of Schistosoma mansoni is expressed in the gonads, and its pharmacological inhibition reduces mitogenic activity and egg production in paired females in vitro. Since Src kinases are important signal transduction proteins it is of interest to unravel the signaling cascades SmTK3 is involved in to understand its cellular role in the gonads.

Methodology and results: Towards this end we established and screened a yeast two-hybrid (Y2H) cDNA library of adult S. mansoni with a bait construct encoding the SH3 (src homology) domain and unique site of SmTK3. Among the binding partners found was a diaphanous homolog (SmDia), which was characterized further. SmDia is a single-copy gene transcribed throughout development with a bias towards male transcription. Its deduced amino acid sequence reveals all diaphanous-characteristic functional domains. Binding studies with truncated SmDia clones identified SmTK3 interaction sites demonstrating that maximal binding efficiency depends on the N-terminal part of the FH1 (formin homology) domain and the inter-domain region of SmDia located upstream of FH1 in combination with the unique site and the SH3 domain of SmTK3, respectively. SmDia also directly interacted with the GTPase SmRho1 of S. mansoni. In situ hybridization experiments finally demonstrated that SmDia, SmRho1, and SmTK3 are transcribed in the gonads of both genders.

Conclusion: These data provide first evidence for the existence of two cooperating pathways involving Rho and Src that bridge at SmDia probably organizing cytoskeletal events in the reproductive organs of a parasite, and beyond that in gonads of eukaryotes. Furthermore, the FH1 and inter domain region of SmDia have been discovered as binding sites for the SH3 and unique site domains of SmTK3, respectively.

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Schematic structure of SmTK3 and expression of the SmTK3-bait proteins in yeast.A: The diagram shows the structure of SmTK3 with the Src-homology domains 2–4 (SH2-SH4), the tyrosine-kinase domain (catalytic domain), and the unique site (unique). The parts of SmTK3 used as baits for Y2H-library screening are shown below the diagram (TK3-SH3 and TK3-US-SH3). B: Western blot analyses of protein lysates of SmTK3-SH3(+/-US) pGBKT9-transformed Y187 yeast-cells to confirm the expression of the bait constructs. Using an anti-Gal4 antibody, bands of the expected sizes for the fusion proteins Gal4 DNA-BD/TK3-US-SH3 (lane 1) and Gal4-DNA-BD/TK3-SH3 (lane 2) were detected. Marker (M): 10 kDa ladder (Gibco BRL).
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pone-0006998-g001: Schematic structure of SmTK3 and expression of the SmTK3-bait proteins in yeast.A: The diagram shows the structure of SmTK3 with the Src-homology domains 2–4 (SH2-SH4), the tyrosine-kinase domain (catalytic domain), and the unique site (unique). The parts of SmTK3 used as baits for Y2H-library screening are shown below the diagram (TK3-SH3 and TK3-US-SH3). B: Western blot analyses of protein lysates of SmTK3-SH3(+/-US) pGBKT9-transformed Y187 yeast-cells to confirm the expression of the bait constructs. Using an anti-Gal4 antibody, bands of the expected sizes for the fusion proteins Gal4 DNA-BD/TK3-US-SH3 (lane 1) and Gal4-DNA-BD/TK3-SH3 (lane 2) were detected. Marker (M): 10 kDa ladder (Gibco BRL).

Mentions: Cloning candidate genes or gene fragments into vectors for Y2H analyses was done as follows. One bait vector containing the 5′ unique site combined with the SH3 domain (611 bp fragment) of SmTK3 was constructed to screen for “downstream” interaction partners [13]. Another bait vector was cloned, which contained the single 185 bp long SH3 domain amplified by the primer combination TK3-SH3-1 (5′-GGAGAATTCGGGCAGTTTGTTGCTTTAC-3′; containing an EcoRI restriction site) and TK3-SH3-2 (5′-TTCGTCGACGGATTCCAAACTGGTAAC-3′; containing a SalI restriction site). This vector was used for subsequent comparative Y2H analyses to investigate the role of the unique site for the binding efficiency of potential interaction partners (Fig. 1A). Each bait fragment was cloned in frame with the Gal4 DNA-BD into pGBKT9.


The formin-homology protein SmDia interacts with the Src kinase SmTK and the GTPase SmRho1 in the gonads of Schistosoma mansoni.

Quack T, Knobloch J, Beckmann S, Vicogne J, Dissous C, Grevelding CG - PLoS ONE (2009)

Schematic structure of SmTK3 and expression of the SmTK3-bait proteins in yeast.A: The diagram shows the structure of SmTK3 with the Src-homology domains 2–4 (SH2-SH4), the tyrosine-kinase domain (catalytic domain), and the unique site (unique). The parts of SmTK3 used as baits for Y2H-library screening are shown below the diagram (TK3-SH3 and TK3-US-SH3). B: Western blot analyses of protein lysates of SmTK3-SH3(+/-US) pGBKT9-transformed Y187 yeast-cells to confirm the expression of the bait constructs. Using an anti-Gal4 antibody, bands of the expected sizes for the fusion proteins Gal4 DNA-BD/TK3-US-SH3 (lane 1) and Gal4-DNA-BD/TK3-SH3 (lane 2) were detected. Marker (M): 10 kDa ladder (Gibco BRL).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2734992&req=5

pone-0006998-g001: Schematic structure of SmTK3 and expression of the SmTK3-bait proteins in yeast.A: The diagram shows the structure of SmTK3 with the Src-homology domains 2–4 (SH2-SH4), the tyrosine-kinase domain (catalytic domain), and the unique site (unique). The parts of SmTK3 used as baits for Y2H-library screening are shown below the diagram (TK3-SH3 and TK3-US-SH3). B: Western blot analyses of protein lysates of SmTK3-SH3(+/-US) pGBKT9-transformed Y187 yeast-cells to confirm the expression of the bait constructs. Using an anti-Gal4 antibody, bands of the expected sizes for the fusion proteins Gal4 DNA-BD/TK3-US-SH3 (lane 1) and Gal4-DNA-BD/TK3-SH3 (lane 2) were detected. Marker (M): 10 kDa ladder (Gibco BRL).
Mentions: Cloning candidate genes or gene fragments into vectors for Y2H analyses was done as follows. One bait vector containing the 5′ unique site combined with the SH3 domain (611 bp fragment) of SmTK3 was constructed to screen for “downstream” interaction partners [13]. Another bait vector was cloned, which contained the single 185 bp long SH3 domain amplified by the primer combination TK3-SH3-1 (5′-GGAGAATTCGGGCAGTTTGTTGCTTTAC-3′; containing an EcoRI restriction site) and TK3-SH3-2 (5′-TTCGTCGACGGATTCCAAACTGGTAAC-3′; containing a SalI restriction site). This vector was used for subsequent comparative Y2H analyses to investigate the role of the unique site for the binding efficiency of potential interaction partners (Fig. 1A). Each bait fragment was cloned in frame with the Gal4 DNA-BD into pGBKT9.

Bottom Line: The Src-like tyrosine-kinase SmTK3 of Schistosoma mansoni is expressed in the gonads, and its pharmacological inhibition reduces mitogenic activity and egg production in paired females in vitro.Among the binding partners found was a diaphanous homolog (SmDia), which was characterized further.Furthermore, the FH1 and inter domain region of SmDia have been discovered as binding sites for the SH3 and unique site domains of SmTK3, respectively.

View Article: PubMed Central - PubMed

Affiliation: Institute for Parasitology, Justus-Liebig-University, Giessen, Germany.

ABSTRACT

Background: Schistosomiasis (bilharzia) is a parasitic disease of worldwide significance affecting human and animals. As schistosome eggs are responsible for pathogenesis, the understanding of processes controlling gonad development might open new perspectives for intervention. The Src-like tyrosine-kinase SmTK3 of Schistosoma mansoni is expressed in the gonads, and its pharmacological inhibition reduces mitogenic activity and egg production in paired females in vitro. Since Src kinases are important signal transduction proteins it is of interest to unravel the signaling cascades SmTK3 is involved in to understand its cellular role in the gonads.

Methodology and results: Towards this end we established and screened a yeast two-hybrid (Y2H) cDNA library of adult S. mansoni with a bait construct encoding the SH3 (src homology) domain and unique site of SmTK3. Among the binding partners found was a diaphanous homolog (SmDia), which was characterized further. SmDia is a single-copy gene transcribed throughout development with a bias towards male transcription. Its deduced amino acid sequence reveals all diaphanous-characteristic functional domains. Binding studies with truncated SmDia clones identified SmTK3 interaction sites demonstrating that maximal binding efficiency depends on the N-terminal part of the FH1 (formin homology) domain and the inter-domain region of SmDia located upstream of FH1 in combination with the unique site and the SH3 domain of SmTK3, respectively. SmDia also directly interacted with the GTPase SmRho1 of S. mansoni. In situ hybridization experiments finally demonstrated that SmDia, SmRho1, and SmTK3 are transcribed in the gonads of both genders.

Conclusion: These data provide first evidence for the existence of two cooperating pathways involving Rho and Src that bridge at SmDia probably organizing cytoskeletal events in the reproductive organs of a parasite, and beyond that in gonads of eukaryotes. Furthermore, the FH1 and inter domain region of SmDia have been discovered as binding sites for the SH3 and unique site domains of SmTK3, respectively.

Show MeSH
Related in: MedlinePlus