Tv-RIO1 - an atypical protein kinase from the parasitic nematode Trichostrongylus vitrinus

Abstract

Background: Protein kinases are key enzymes that regulate a wide range of cellular processes, including cell-cycle progression, transcription, DNA replication and metabolic functions. These enzymes catalyse the transfer of phosphates to serine, threonine and tyrosine residues, thus playing functional roles in reversible protein phosphorylation. There are two main groups, namely eukaryotic protein kinases (ePKs) and atypical protein kinases (aPKs); RIO kinases belong to the latter group. While there is some information about RIO kinases and their roles in animals, nothing is known about them in parasites. This is the first study to characterise a RIO1 kinase from any parasite.

Results: A full-length cDNA (Tv-rio-1) encoding a RIO1 protein kinase (Tv-RIO1) was isolated from the economically important parasitic nematode Trichostrongylus vitrinus (Order Strongylida). The uninterrupted open reading frame (ORF) of 1476 nucleotides encoded a protein of 491 amino acids, containing the characteristic RIO1 motif LVHADLSEYNTL. Tv-rio-1 was transcribed at the highest level in the third-stage larva (L3), and a higher level in adult females than in males. Comparison with homologues from other organisms showed that protein Tv-RIO1 had significant homology to related proteins from a range of metazoans and plants. Amino acid sequence identity was most pronounced in the ATP-binding motif, active site and metal binding loop. Phylogenetic analyses of selected amino acid sequence data revealed Tv-RIO1 to be most closely related to the proteins in the species of Caenorhabditis. A structural model of Tv-RIO1 was constructed and compared with the published crystal structure of RIO1 of Archaeoglobus fulgidus (Af-Rio1).

Conclusion: This study provides the first insights into the RIO1 protein kinases of nematodes, and a foundation for further investigations into the biochemical and functional roles of this molecule in biological processes in parasitic nematodes.

Figure 1

Full-length cDNA sequence of the cDNA (Tv-rio-1) encoding the protein Tv-RIO1 from Trichostrongylus vitrinus, and its predicted amino acid sequence. The nucleotide sequence determined from the original EST TVf09_H06 (accession no. NP_491102.2; [18]) is in bold text. The untranslated regions are in lower case, and protein-coding nucleotide sequence is in upper case. ATG and TAG (asterisk) are the inferred translation initiation and termination signals, respectively; the putative polyadenylation signal sequence is underlined. The amino acid residues representing the RIO1 protein kinase signature LVHADLSEYNTL (PS01245) are shaded.

Figure 2

An alignment of the inferred amino acid sequences of RIO1 proteins from Trichostrongylus vitrinus and other nine selected species. These nine species include Caenorhabditis elegans (accession no. AAC17564), C. briggsae (CAE60574), Brugia malayi (EDP30009), Homo sapiens (NP_113668), Mus musculus (NP_077204), Danio rerio (NP_998160), Drosophila melanogaster (NP_648489), Arabidopsis thaliana (NP_180071) and Schizosaccharomyces pombe (CAA15723). Amino acids predicted to be involved in the ATP binding motif and active sites are boxed. Identical amino acids are marked with asterisks. Predicted subdomains I-XI (see Fig. 1B; [7]) are marked above the alignment. Alpha-helices A-I (shaded) or beta-sheet structures (underlined) [1] are indicated. Flexible loop, hinge and metal-binding loop were identified (see Fig. 1D in [40]). Dashes indicate gaps in the sequence, included for alignment purposes.

Figure 3

Genetic relationship of Tv-RIO1 with homologues from a range of organisms. Neighbour-joining tree displays the relationship of Tv-RIO1 from Trichostrongylus vitrinus with RIO1 protein kinases from 16 species representing different phyla. These species are Caenorhabditis elegans, C. briggsae, Brugia malayi (nematodes); Arabidopsis thaliana, Nicotiana tabacum, Oryza sativa (plants); Homo sapiens, Macaca fascicularis, Pan troglodytes, Pongo pygmaeus (primates); Canis familiaris, Rattus norvegicus, Mus musculus (other mammals); Danio rerio (zebrafish); Drosophila melanogaster (vinegar fly) and Dictyostelium discoideum (slime mould). Accession numbers identify individual sequences representing individual species. Schizosaccharomyces pombe (fission yeast) (accession no. CAA15723) represents the outgroup. Numbers above and below the branches are the bootstrap values (%) obtained using the neighbour-joining and maximum parsimony methods, respectively.

Figure 4

Homology model of the Tv-RIO1 protein from Trichostrongylus vitrinus. The loop in red indicates the region of dissimilarity between Tv-RIO1 and Af-RIO1 from Archaeoglobus fulgidus. The P-loop required for the binding to phosphate is shown in blue. Catalytic residues (Asn 281, Asp 276 and Asp 293) and the highly conserved Tyr 280 are indicated by yellow sticks.

Figure 5

Transcription of Tv-rio-1 in Trichostrongylus vitrinus. Transcriptional profile of Tv-rio-1 in different developmental stages [third – (L3) and fourth – (L4) stage larvae] and genders [females (F) and males (M)] of Trichostrongylus vitrinus, determined by real-time PCR analysis. Data shown are mean values (± standard error of the mean) derived from three replicates in repeat experiments. Relative transcription was calculated by normalization of the raw data, followed by the determination of abundance relative to a calibrator. Quantification of the cDNA representing Tv-rio1 in each sample was normalised, using part of the large subunit (28S) of the nuclear ribosomal RNA gene as an endogenous control.

Figure 6

Probabilistic functional gene network analysis for Caenorhabditis elegans gene M01B12.5. (using the recommended cut-off value; [35]).