Conservation of protein kinase a catalytic subunit sequences in the schistosome pathogens of humans

Abstract

cAMP-dependent protein kinases (PKAs) are central mediators of cAMP signaling in eukaryotic cells. Previously we identified a cDNA which encodes for a PKA catalytic subunit (PKA-C) in Schistosoma mansoni (SmPKA-C) that is required for adult schistosome viability in vitro. As such, SmPKA-C could potentially represent a novel schistosome chemotherapeutic target. Here we sought to identify PKA-C subunit orthologues in the other medically important schistosome species, Schistosoma haematobium and Schistosoma japonicum, to determine the degree to which this potential target is conserved and could therefore be exploited for the treatment of all forms of schistosomiasis. We report the identification of PKA-C subunit orthologues in S. haematobium and S. japonicum (ShPKA-C and SjPKA-C, respectively) and show that PKA-C orthologues are highly conserved in the Schistosoma, with over 99% amino acid sequence identity shared among the three human pathogens we examined. Furthermore, we show that the recently published Schistosoma mansoni and S. japonicum genomes contain sequences encoding for several putative PKA substrates with homology to those found in Homo sapiens, Caenorhabditis elegans, and Saccharomyces cerevisiae.

Figure 1

Amino acid alignment of PKA-C subunit sequences from Schistosoma. Amino acid sequences of PKA-C subunits from Schistosoma mansoni (SmPKA-C; GQ168377), S. haematobium (ShPKA-C; GU116484), S. japonicum (SjPKA-C; GU130533) and Homo sapiens PKA-Cα (HsPKA-Ca; P17612), H. sapiens PKA-Cβ (HsPKA-Cb; P22694), and H. sapiens PKA-Cγ (HsPKA-Cg; P22612) were aligned using the ClustalW algorithm. Underlines indicate conserved amino acid sequences as follows: 1, kinase domain (Phe43 – Phe297); 2, ATP-binding site (Gly50 - Val57); 3, Ser/Thr active site (Arg165 - Asn171); 4, conserved autophosphorylation site (Thr196); 5, conserved sequence motif for PKA regulatory subunit binding (Leu198 - Tyr204).

Figure 2

Phylogenetic analysis of PKA-C cDNA nucleotide and amino acid sequences from schistosomes and other species. A, Phylogenetic analysis of PKA-C cDNA nucleotide sequences of schistosomes. PKA-C ORF sequences were aligned using the ClustalW algorithm and the tree constructed using the Maximum Parsimony method. The bootstrap consensus tree inferred from 500 replicates is shown. The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test is shown next to the branch. The tree is drawn to scale, with branch lengths calculated using the average pathway method (Tamura, et al., 2007) and are in the units of the number of changes over the whole sequence. There were a total of 1053 positions in the final alignment, out of which 52 were parsimony informative. Nucleotide sequences of the predicted open reading frames of the schistosome PKA-C subunits are labeled as follows: ShPKA-C, S. haematobium PKA-C (GU116484) SmPKA-C, S. mansoni PKA-C (GQ168377); SjPKA-C, S. japonicum PKA-C (GU130533). The H. sapiens PKA-Cα nucleotide sequence (BC108259), labeled HsPKA-Ca, is included for comparison. B, Phylogenetic analysis of PKA-C amino acid sequences from schistosomes and other species. Sequences were aligned using the ClustalW algorithm and the tree constructed using the Maximum Parsimony method. The bootstrap consensus tree inferred from 500 replicates is shown. The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test is shown next to the branch. The tree is drawn to scale, with branch lengths calculated using the average pathway method (Tamura, et al., 2007) and are in the units of the number of changes over the whole sequence. There were a total of 350 positions in the final alignment, out of which 93 were parsimony informative. Amino acid sequences are labeled as follows: HsPKA-Ca, Homo sapiens PKA-Cα (P17612); HsPKA-Cb, H. sapiens PKA-Cβ (P22694); HsPKA-Cg, H. sapiens PKA-Cγ (P22612); MmPKA-Ca, Mus musculus PKA-Cα (P05132); MmPKA-Cb, M. musculus PKA-Cβ (P68181); RnPKA-Ca, Rattus norvegicus PKA-Cα (P27791); RnPKA-Cb, R. norvegicus PKA-Cγ (P68182); AcPKA-C, Ancylostoma caninum PKA-C (U15983); PhcPKA-C, Pediculus humanus corporis PKA-C (XM_0024273690); CqPKA-C, Culex quinquefasciatus PKA-C (XM_001868382).