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Toward understanding the functional role of Ss-RIOK-1, a RIO protein kinase-encoding gene of Strongyloides stercoralis.

Yuan W, Lok JB, Stoltzfus JD, Gasser RB, Fang F, Lei WQ, Fang R, Zhou YQ, Zhao JL, Hu M - PLoS Negl Trop Dis (2014)

Bottom Line: Recombinant Ss-RIOK-1 is an active kinase, capable of both phosphorylation and auto-phosphorylation.Patterns of transcriptional reporter expression in transgenic S. stercoralis larvae indicated that Ss-RIOK-1 is expressed in neurons of the head, body and tail as well as in pharynx and hypodermis.The characterization of the molecular and the temporal and spatial expression patterns of the encoding gene provide first clues as to functions of RIOKs in the biological processes of parasitic nematodes.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.

ABSTRACT

Background: Some studies of Saccharomyces cerevisiae and mammals have shown that RIO protein kinases (RIOKs) are involved in ribosome biogenesis, cell cycle progression and development. However, there is a paucity of information on their functions in parasitic nematodes. We aimed to investigate the function of RIOK-1 encoding gene from Strongyloides stercoralis, a nematode parasitizing humans and dogs.

Methodology/principal findings: The RIOK-1 protein-encoding gene Ss-riok-1 was characterized from S. stercoralis. The full-length cDNA, gDNA and putative promoter region of Ss-riok-1 were isolated and sequenced. The cDNA comprises 1,828 bp, including a 377 bp 5'-UTR, a 17 bp 3'-UTR and a 1,434 bp ORF encoding a protein of 477 amino acids containing a RIOK-1 signature motif. The genomic sequence of the Ss-riok-1 coding region is 1,636 bp in length and has three exons and two introns. The putative promoter region comprises 4,280 bp and contains conserved promoter elements, including four CAAT boxes, 12 GATA boxes, eight E-boxes (CANNTG) and 38 TATA boxes. The Ss-riok-1 gene is transcribed throughout all developmental stages with the highest transcript abundance in the infective third-stage larva (iL3). Recombinant Ss-RIOK-1 is an active kinase, capable of both phosphorylation and auto-phosphorylation. Patterns of transcriptional reporter expression in transgenic S. stercoralis larvae indicated that Ss-RIOK-1 is expressed in neurons of the head, body and tail as well as in pharynx and hypodermis.

Conclusions/significance: The characterization of the molecular and the temporal and spatial expression patterns of the encoding gene provide first clues as to functions of RIOKs in the biological processes of parasitic nematodes.

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Related in: MedlinePlus

Autophosphorylation and phosphorylation activity of recombinant GST-Ss-RIOK-1.A, Western-blot of GST-Ss-RIOK-1 (89 kDa) and GST (28 kDa) are immunoprecipitated with GST antibody. B, Kinase assay showing autophosphorylation and phosphorylation activities of recombinant GST-Ss-RIOK-1 (89 kDa). Substrate in phosphorylation assay is MBP (18 kDa). Amounts of each protein in the reaction are indicated in the labels. Purified GST constitutes the negative control.
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pntd-0003062-g005: Autophosphorylation and phosphorylation activity of recombinant GST-Ss-RIOK-1.A, Western-blot of GST-Ss-RIOK-1 (89 kDa) and GST (28 kDa) are immunoprecipitated with GST antibody. B, Kinase assay showing autophosphorylation and phosphorylation activities of recombinant GST-Ss-RIOK-1 (89 kDa). Substrate in phosphorylation assay is MBP (18 kDa). Amounts of each protein in the reaction are indicated in the labels. Purified GST constitutes the negative control.

Mentions: The activities of many protein kinases include phosphorylation and auto-phosphorylation. It is reported that RIOK-1 could also phosphorylate the common protein kinase substrate MBP as well as RIOK-1 itself [49]. To assess the kinase activity of Ss-RIOK-1, recombinant GST-Ss-RIOK-1 with a GST tag (designated GST-Ss-RIOK-1) was expressed in E. coli (Fig. 5A). Purified recombinant GST-Ss-RIOK-1 incubated with [γ32P] ATP only or in the presence of MBP showed radioactive signals associated with the Ss-RIOK-1 and MBP, respectively, indicating that the GST-Ss-RIOK-1 is capable of both phosphorylation and auto-phosphorylation (Fig. 5B).


Toward understanding the functional role of Ss-RIOK-1, a RIO protein kinase-encoding gene of Strongyloides stercoralis.

Yuan W, Lok JB, Stoltzfus JD, Gasser RB, Fang F, Lei WQ, Fang R, Zhou YQ, Zhao JL, Hu M - PLoS Negl Trop Dis (2014)

Autophosphorylation and phosphorylation activity of recombinant GST-Ss-RIOK-1.A, Western-blot of GST-Ss-RIOK-1 (89 kDa) and GST (28 kDa) are immunoprecipitated with GST antibody. B, Kinase assay showing autophosphorylation and phosphorylation activities of recombinant GST-Ss-RIOK-1 (89 kDa). Substrate in phosphorylation assay is MBP (18 kDa). Amounts of each protein in the reaction are indicated in the labels. Purified GST constitutes the negative control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125297&req=5

pntd-0003062-g005: Autophosphorylation and phosphorylation activity of recombinant GST-Ss-RIOK-1.A, Western-blot of GST-Ss-RIOK-1 (89 kDa) and GST (28 kDa) are immunoprecipitated with GST antibody. B, Kinase assay showing autophosphorylation and phosphorylation activities of recombinant GST-Ss-RIOK-1 (89 kDa). Substrate in phosphorylation assay is MBP (18 kDa). Amounts of each protein in the reaction are indicated in the labels. Purified GST constitutes the negative control.
Mentions: The activities of many protein kinases include phosphorylation and auto-phosphorylation. It is reported that RIOK-1 could also phosphorylate the common protein kinase substrate MBP as well as RIOK-1 itself [49]. To assess the kinase activity of Ss-RIOK-1, recombinant GST-Ss-RIOK-1 with a GST tag (designated GST-Ss-RIOK-1) was expressed in E. coli (Fig. 5A). Purified recombinant GST-Ss-RIOK-1 incubated with [γ32P] ATP only or in the presence of MBP showed radioactive signals associated with the Ss-RIOK-1 and MBP, respectively, indicating that the GST-Ss-RIOK-1 is capable of both phosphorylation and auto-phosphorylation (Fig. 5B).

Bottom Line: Recombinant Ss-RIOK-1 is an active kinase, capable of both phosphorylation and auto-phosphorylation.Patterns of transcriptional reporter expression in transgenic S. stercoralis larvae indicated that Ss-RIOK-1 is expressed in neurons of the head, body and tail as well as in pharynx and hypodermis.The characterization of the molecular and the temporal and spatial expression patterns of the encoding gene provide first clues as to functions of RIOKs in the biological processes of parasitic nematodes.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.

ABSTRACT

Background: Some studies of Saccharomyces cerevisiae and mammals have shown that RIO protein kinases (RIOKs) are involved in ribosome biogenesis, cell cycle progression and development. However, there is a paucity of information on their functions in parasitic nematodes. We aimed to investigate the function of RIOK-1 encoding gene from Strongyloides stercoralis, a nematode parasitizing humans and dogs.

Methodology/principal findings: The RIOK-1 protein-encoding gene Ss-riok-1 was characterized from S. stercoralis. The full-length cDNA, gDNA and putative promoter region of Ss-riok-1 were isolated and sequenced. The cDNA comprises 1,828 bp, including a 377 bp 5'-UTR, a 17 bp 3'-UTR and a 1,434 bp ORF encoding a protein of 477 amino acids containing a RIOK-1 signature motif. The genomic sequence of the Ss-riok-1 coding region is 1,636 bp in length and has three exons and two introns. The putative promoter region comprises 4,280 bp and contains conserved promoter elements, including four CAAT boxes, 12 GATA boxes, eight E-boxes (CANNTG) and 38 TATA boxes. The Ss-riok-1 gene is transcribed throughout all developmental stages with the highest transcript abundance in the infective third-stage larva (iL3). Recombinant Ss-RIOK-1 is an active kinase, capable of both phosphorylation and auto-phosphorylation. Patterns of transcriptional reporter expression in transgenic S. stercoralis larvae indicated that Ss-RIOK-1 is expressed in neurons of the head, body and tail as well as in pharynx and hypodermis.

Conclusions/significance: The characterization of the molecular and the temporal and spatial expression patterns of the encoding gene provide first clues as to functions of RIOKs in the biological processes of parasitic nematodes.

Show MeSH
Related in: MedlinePlus