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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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Comparative liquid β-Gal assays to determine the relative binding strengths of potential SmTK3 interaction partners.Comparative β-Gal liquid assays were performed to determine the relative binding strength of the identified interaction partners (n = 6). A representative member of each clone group was re-transformed in yeast cells (AH109) together with TK3-SH3 (light grey) or TK3-US-SH3 (dark grey). The tested clones were (from left to right): diaphanous (SmDia), the eukaryotic translation initiation factor (eIF4γ2b), the BAF60 subunit of the SWI/SNF complex (SWI/SNF-BAF60), the YME1-like metalloprotease (YME1-homologue), the mRNA (guanine-7) methyl transferase (RNA methyl TF), the quinolinate phosphoribosyl-transferase (phosphoribosyl TF), the SH3 binding protein (SH3 domain BP), the Smad protein (Smad2/3), and pericentrin B. As negative control, yeast cells were transformed with TK3-US-SH3 only (bait only).
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pone-0006998-g003: Comparative liquid β-Gal assays to determine the relative binding strengths of potential SmTK3 interaction partners.Comparative β-Gal liquid assays were performed to determine the relative binding strength of the identified interaction partners (n = 6). A representative member of each clone group was re-transformed in yeast cells (AH109) together with TK3-SH3 (light grey) or TK3-US-SH3 (dark grey). The tested clones were (from left to right): diaphanous (SmDia), the eukaryotic translation initiation factor (eIF4γ2b), the BAF60 subunit of the SWI/SNF complex (SWI/SNF-BAF60), the YME1-like metalloprotease (YME1-homologue), the mRNA (guanine-7) methyl transferase (RNA methyl TF), the quinolinate phosphoribosyl-transferase (phosphoribosyl TF), the SH3 binding protein (SH3 domain BP), the Smad protein (Smad2/3), and pericentrin B. As negative control, yeast cells were transformed with TK3-US-SH3 only (bait only).

Mentions: To investigate the relative binding strengths of the potential interaction partners, representative members of each clone group were re-transformed in yeast cells together with TK3-SH3 or TK3-US-SH3 for comparative liquid β-Gal assays (Fig. 3). This quantitative test again confirmed interaction, and, in addition, demonstrated the strongest binding of TK3-SH3 with BAF60 and diaphanous. In both cases, binding was enhanced in the presence of the unique site. This applied also to the representatives of the other clone groups except Smad 2/3 (group H), where the SH3 domain alone led to a stronger interaction. In two cases, the YME1-like metalloprotease (group D) and Qprt (group F), the unique site seemed to be exclusively responsible for the binding activity. As expected, no β-Gal activity was measured when control yeast cells were transformed with the bait plasmids only. These findings correspond to previous reports suggesting that the unique site may reinforce binding of TKs to their substrates [27], or may even act itself as a binding domain [28].


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)

Comparative liquid β-Gal assays to determine the relative binding strengths of potential SmTK3 interaction partners.Comparative β-Gal liquid assays were performed to determine the relative binding strength of the identified interaction partners (n = 6). A representative member of each clone group was re-transformed in yeast cells (AH109) together with TK3-SH3 (light grey) or TK3-US-SH3 (dark grey). The tested clones were (from left to right): diaphanous (SmDia), the eukaryotic translation initiation factor (eIF4γ2b), the BAF60 subunit of the SWI/SNF complex (SWI/SNF-BAF60), the YME1-like metalloprotease (YME1-homologue), the mRNA (guanine-7) methyl transferase (RNA methyl TF), the quinolinate phosphoribosyl-transferase (phosphoribosyl TF), the SH3 binding protein (SH3 domain BP), the Smad protein (Smad2/3), and pericentrin B. As negative control, yeast cells were transformed with TK3-US-SH3 only (bait only).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2734992&req=5

pone-0006998-g003: Comparative liquid β-Gal assays to determine the relative binding strengths of potential SmTK3 interaction partners.Comparative β-Gal liquid assays were performed to determine the relative binding strength of the identified interaction partners (n = 6). A representative member of each clone group was re-transformed in yeast cells (AH109) together with TK3-SH3 (light grey) or TK3-US-SH3 (dark grey). The tested clones were (from left to right): diaphanous (SmDia), the eukaryotic translation initiation factor (eIF4γ2b), the BAF60 subunit of the SWI/SNF complex (SWI/SNF-BAF60), the YME1-like metalloprotease (YME1-homologue), the mRNA (guanine-7) methyl transferase (RNA methyl TF), the quinolinate phosphoribosyl-transferase (phosphoribosyl TF), the SH3 binding protein (SH3 domain BP), the Smad protein (Smad2/3), and pericentrin B. As negative control, yeast cells were transformed with TK3-US-SH3 only (bait only).
Mentions: To investigate the relative binding strengths of the potential interaction partners, representative members of each clone group were re-transformed in yeast cells together with TK3-SH3 or TK3-US-SH3 for comparative liquid β-Gal assays (Fig. 3). This quantitative test again confirmed interaction, and, in addition, demonstrated the strongest binding of TK3-SH3 with BAF60 and diaphanous. In both cases, binding was enhanced in the presence of the unique site. This applied also to the representatives of the other clone groups except Smad 2/3 (group H), where the SH3 domain alone led to a stronger interaction. In two cases, the YME1-like metalloprotease (group D) and Qprt (group F), the unique site seemed to be exclusively responsible for the binding activity. As expected, no β-Gal activity was measured when control yeast cells were transformed with the bait plasmids only. These findings correspond to previous reports suggesting that the unique site may reinforce binding of TKs to their substrates [27], or may even act itself as a binding domain [28].

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