Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote

Abstract

Background: Malaria, caused by the parasitic protist Plasmodium falciparum, represents a major public health problem in the developing world. The P. falciparum genome has been sequenced, which provides new opportunities for the identification of novel drug targets. Eukaryotic protein kinases (ePKs) form a large family of enzymes with crucial roles in most cellular processes; hence malarial ePKS represent potential drug targets. We report an exhaustive analysis of the P. falciparum genomic database (PlasmoDB) aimed at identifying and classifying all ePKs in this organism.

Results: Using a variety of bioinformatics tools, we identified 65 malarial ePK sequences and constructed a phylogenetic tree to position these sequences relative to the seven established ePK groups. Predominant features of the tree were: (i) that several malarial sequences did not cluster within any of the known ePK groups; (ii) that the CMGC group, whose members are usually involved in the control of cell proliferation, had the highest number of malarial ePKs; and (iii) that no malarial ePK clustered with the tyrosine kinase (TyrK) or STE groups, pointing to the absence of three-component MAPK modules in the parasite. A novel family of 20 ePK-related sequences was identified and called FIKK, on the basis of a conserved amino acid motif. The FIKK family seems restricted to Apicomplexa, with 20 members in P. falciparum and just one member in some other Apicomplexan species.

Conclusion: The considerable phylogenetic distance between Apicomplexa and other Eukaryotes is reflected by profound divergences between the kinome of malaria parasites and that of yeast or mammalian cells.

Figure 1

Phylogenetic distance between malaria parasites and the organisms used as model Eukaryotes. With the exception of the plant Arabidopsis, the organisms whose kinome has been characterised (yeast, worms, Drosophila and human), all belong to the Opisthokonta lineage, which is vastly distant from the Alveolata branch which include the Apicomplexa. Adapted from Badlauf, S (2003), with permission (Copyright 2003 AAAS).

Figure 2

The P. falciparum kinome. Phylogenetic tree of ePKs from P. falciparum. The tree was compiled using conserved portions of aligned sequences using a protein distance matrix method (see Additional file 1 for the alignment). All major groupings discussed were observed in the 100 replicate bootstrap tree (not shown). Branches with bootstrap values >70 are shown in red and >40 in blue. The scale bar represents 0.1 mutational changes per residue (10 PAM units). 65 sequences from P. falciparum are shown (in red characters), together with representative members of major subgroups of human kinases (in black characters). The P. falciparum sequences are labelled with their identifier in the PlasmoDB database and, where applicable, with the published name of the enzymes. The human sequences are labeled with HUGO gene names.

Figure 3

A three-species tree of the CMGC group. Phylogenetic tree showing members of the CMGC group of protein kinases from P. falciparum, yeast and human. The tree was compiled using conserved portions of aligned sequences using a protein distance matrix method; the tree shown is a consensus tree built from 100 bootstrap replicates. Branches with bootstrap values >70 are shown in red and >40 in blue. The scale bar represents 0.1 mutational changes per residue (10 PAM units). The P. falciparum sequences are identified by with their identifier in the PlasmoDB database and, where applicable, with the published name of the enzymes. The human sequences are labeled (black) with HUGO gene names (except for sk466, which is a numerical designation taken from Manning et al. (2002), and the yeast sequences (blue) identified according to the catalogue in Hunter and Plowman (1997).

Figure 4

Comparative primary structure of FIKKs and typical ePKs. The eleven subdomains of the protein kinase catalytic domain are indicated in the central bar. The residues which are conserved in most ePKs (see legend to Table 1 for details) are indicated at the top. The corresponding residues in FIKKs are indicated under the bar, together with some of the motifs with which they are associated and which are conserved in all FIKK family members.

Figure 5

Alignment of four representative sequences of the FIKK family with a typical ePK (PfPK5 [MAL13P1.279], a CDK homologue). Asterisks indicate those residues that are invariant in all 20 FIKK sequences.

Figure 6

A tree of the FIKK family. Phylogenetic tree of FIKKs from P. falciparum. The tree was compiled using conserved portions of aligned sequences (see Additional file 3) using a protein distance matrix method. The scale bar represents 0.1 mutational changes per residues (10 PAM units). Bootstrap values over 75 are shown. The bottom panel shows a map of one of the telomeric and subtelomeric regions of chromosome 9 obtained from the PlasmoDB website. The location of genes encoding proteins of the var/PfEMP1 (Duffy et al., 2003), rifin (Kyes et al., 1999) and FIKK (this study) families is indicated.

Figure 7

P. falciparum ePKs and related proteins, and stage-specificity of their expression. PlasmoDB gene identifiers are indicated in the left column, followed by the published names where applicable. Identifiers of enzymes belonging to defined ePK groups appear in color (see the inset for color codes). Microarray data from the Le Roch et al. and Bozdech et al. studies available on PlasmoDB, were compiled to produce the third column. Genes were arranged in function of the timing of their expression according to Bozdech et al., to illustrate the fact essentially all of them are expressed in a regulated way during erythrocytic schizogony, and that this process involves sequential but overlapping expression of most kinases in the genome. The phaseogram (data generated by Bozdech et al. and available on the PlasmoDB website) represent the relative abundance of mRNAs throughout the erythrocytic asexual cycle, measured by two-colour competitive hybridisation between total RNA from each time point and a reference pool of total RNA from all time points (48 time points, i.e. one per hour during the 48 hours of the asexual cycle, starting one hour post invasion). The phaseogram shows the red/green colorimetric representation of the gene expression ratio for each oligonucleotide. Green: negative ratio (no expression), red: positive ratio (expression); grey or white: no data. See Bozdech et al. (2003) and PlasmoDB for details. To the right of the phaseogram, the presence or absence of mRNA in samples from merozoites (M), gametocytes (G) and sporozoites (S) is indicated by red boxes (data generated by Le Roch et al.). Where only one of the two synchronised merozoite population gave a signal, the M box is colored in orange (see Le Roch et al. 2003 for details). Columns to the right indicate those molecules which, according to the gene prediction algorithm used in PlasmoDB, possess a putative apicoplast or mitochondrion targeting sequence (see text for details).