BACKGROUND
modulation of protein phosphorylation through the antagonistic effects of protein kinases and protein phosphatases is a major regulatory mechanism of most cellular processes.
dysregulation of protein phosphorylation in human cells plays a major role in many diseases such as cancers and neurodegenerative disorders  <cit> .
this has prompted the search for drugs targeting protein kinases, an endeavour which led in  <dig> to the commercialisation of gleevec, the first protein kinase inhibitor used as a drug for human disease  <cit> .
additional molecules targeting protein kinases are in clinical trial  <cit> , and significant developments in this field are expected in the next few years.
some of the most devastating infectious diseases are caused by protists such as malaria parasites and trypanosomatids: hence, about half the global population lives in malarious areas, with 10% of the world population contracting the disease each year, which results in 1– <dig> million annual deaths.
the essential role played by eukaryotic protein kinases  in crucial cellular functions makes them attractive potential targets for drugs against such eukaryotic infectious agents  <cit> .
malaria parasites have a complex life cycle.
infection of human beings by plasmodium falciparum, the species responsible for the lethal form of human malaria, begins with the bite of an infected anopheles mosquito, which delivers sporozoites into the bloodstream.
these cells establish an infection inside hepatocytes, where they undergo an intense multiplication generating several thousand merozoites, a process called exo-erythrocytic schizogony.
the merozoites invade erythrocytes, where they also undergo schizogony, the process that is responsible for malaria pathogenesis.
some merozoites, however, arrest the cell cycle and differentiate into male or female gametocytes, which are infective to the mosquito.
once ingested by the insect, the gametocytes develop into gametes  and fuse into a zygote.
further development in the mosquito involves a process of sporogony, producing sporozoites that accumulate in the salivary glands and are now ready to infect a new human host .
the observation that many parasitic epks display profound structural and functional divergences from their counterparts in their vertebrate hosts  <cit>  suggests that specific inhibition is an attainable goal.
the availability of plasmodb, a genomic database for plasmodium falciparum  <cit> , now permits a systematic analysis of the entire complement of epks encoded in the genome  of this pathogen, an important milestone both in our understanding of plasmodium biology and in the definition of potential novel drug targets.
prior to the genomic era, the initial classification system of hanks and quinn  <cit>  distributed epks into four major groups:
• the cyclic-nucleotide- and calcium/phospholipid-dependent kinases ;
• the cmgc group, constituted of the cyclin-dependent- , mitogen-activated- , glycogen-synthase-  and cdk-like kinases;
• the calmodulin-dependent kinases , and
• the tyrosine kinases .
epks that did not clearly fit into any of these groups were placed into the opk  group.
the primary structure of all enzymes in these groups conform to the model described by hanks, in which the catalytic domain is subdivided into eleven subdomains, which can be aligned across all groups.
in addition to the "typical" epks, several enzymes possessing protein kinase activity, but which are unrelated  to epks at the primary structure level, have been identified and termed "atypical protein kinases" .
these include phosphatidyl-inositol 3' kinase , dna-dependent protein kinase, and members of pyruvate dehydrogenase kinase family.
exhaustive analyses of the kinome of some model organisms have now been published.
the kinome of s. cerevisiae contains  <dig> epks  <cit> , and the genomes of d. melanogaster, c. elegans and h. sapiens comprise  <dig>   <dig> and 510– <dig> epk-coding genes, respectively  <cit> .
on the basis of this wealth of new data, three additional major epk groups were recognized  group;
• the ste group, which includes many enzymes functioning in mapk pathways, although the mapks themselves belong to the cmgc group ;
• the tyrosine kinase-like  group, which, as its name indicates, includes enzymes that are related to those in the tyrk group, although they are serine-threonine protein kinases.
furthermore, a description of the  <dig> non-receptor serine/threonine protein kinases of the plant arabidopsis thaliana has recently been published  <cit> .
comparative examination of this and previously available kinomes has demonstrated that members of all major epk groups can be found in yeast, worms, insects, mammals and plants, with the exception of tyrks, which are not found in yeast.
that most tyrks function in hormone-response receptor-linked pathways suggests that this family arose as an adaptation to the needs for intercellular communication in multicellular organisms.
it has however been reported recently that a few unicellular eukaryotes  possess putative tyrk family members  <cit> .
despite the fact that most serine-threonine epks groups are found in all eukaryotes, indicating that their appearance occurred early in evolution, each of the kinomes has nevertheless its specificities.
a striking feature in this respect is the considerable extension of some epk families in some organisms but not in others.
for example, yeast and drosophila have  <dig> and  <dig> members of the casein kinase  <dig>  group respectively, whereas the c. elegans genome encodes  <dig> ck1-related genes.
with the exception of the plant a. thaliana, all eukaryotes whose kinome has been characterised, from yeast to man, belong to the opisthokonta phylogenetic group.
as depicted in fig.
<dig>  this lineage represents only one small branch of the eukaryotic tree.
several eukaryotes of high medical importance, such as malaria parasites  or trypanosomes , belong to phylogenetic groups that are vastly distant from both the opisthokonta and planta branches  <cit> .
this is reflected by many profound peculiarities in their basic biology .
divergences from model eukaryotes can also be expected not only at the level of individual protein kinases of these organisms , but at the level of their kinome as well.
as is documented below, our analysis of the p. falciparum kinome confirms this prediction.
RESULTS
overview of the tree
<dig> sequences related to epks were retrieved from plasmodb and used to construct a phylogenetic tree as described in the methods section .
the tree of the p. falciparum kinome  indicates that although the parasite possesses enzymes belonging to most of the major serine/threonine kinase groups, as described in the following paragraphs, several enzymes do not cluster with any of these groups.
ck <dig> group
only one malarial kinase, the previously described pfck <dig>   <cit> , clearly falls within this group, which is vastly expanded in some other kinomes .
agc group
five malarial kinases cluster within this group, three of which have been characterized: the camp-dependent pfpka   <cit> , the cgmp-dependent pfpkg   <cit> , and pfpkb   <cit> , an enzyme displaying maximal similarity to akt/pkb.
in other eukaryotes, pkb functions in the pi3k-dependent pathway; a pi3k kinase homologue is present in the p. falciparum genome .
two additional sequences  form a separate cluster attached to the base of the agc branch.
there appears to be no clear member of the pkc subfamily.
camk group
the main branch of the tree that contains the human camks also contains  <dig> pfepks, which underlines the importance of calcium signalling in the parasite  <cit> .
a tight cluster is formed by five of these enzymes, which share the overall structure of the calcium-dependent protein kinases  found in plants and ciliates but not in metazoans.
cdpks are characterised by the presence of a kinase catalytic domain located on the same polypeptide as four ef-hand calcium-binding domains.
four of these enzymes have been described previously: pfcdpk <dig>   <cit> , pfcdpk <dig>   <cit> , pfcdkp <dig>   <cit>  and more recently pfcdpk <dig> .
the latter enzyme is expressed in sexual stages and was shown to be essential for development of the parasite in the mosquito, through mediating cell cycle resumption during male gametocyte exflagellation  <cit> .
a fifth cdpk , which like the four cited above possesses four ef-hand motifs, has been discovered in the present study.
pf11_ <dig> appears to be related to cdpks, but contains only one ef-hand motif.
pfpk <dig>  constitutes a sister branch to the cdpk group.
this enzyme was previously characterized as being related to the camk family  <cit> , and has no ef-hand domain.
no malarial kinase clusters closely with the mammalian camks used to anchor the tree.
six other sequences, however, form a sister branch to the cluster that contains the cdpks; only one of these six sequences  possesses an ef-hand domain.
the camk activity described  <cit>  as crucial for ookinete development in the mosquito vector  is likely to be associated with one of the enzymes in this group.
cmgc group
eighteen malarial kinases cluster within this group, which makes it the most prominent group in the plasmodium kinome.
interestingly, in other eukaryotic systems a majority of cmgc kinases are involved in the control of cell proliferation and development, and their relative abundance in the p. falciparum kinome may reflect the variety of successive proliferative and non-proliferative stages which constitute the life cycle of malaria parasites.
six enzymes are related to the cyclin-dependent kinase family,  <dig> of which were identified previously , the last one  having been discovered during the present analysis.
two previously characterised mitogen-activated protein kinases , pfmap- <dig>   <cit>  and pfmap- <dig>   <cit> , cluster together with a member of the mapk family, as expected.
two enzymes, pfpk <dig>   <cit>  and pfcrk- <dig>  , display features of both cdks and mapks according to blastp analysis.
their position either in a cluster  that is intermediate between the cdk and the mapk groups, or in a cluster  at the base of the cdk/mapk/gsk <dig> branch, is consistent with these early observations.
three gsk3-related kinases, two of which  have been characterised previously  <cit> , form a cluster within the cmgc group.
four additional enzymes form another cluster that includes human clk <dig>  one of which is a previously described lammer-related kinase   <cit> .
the complexity of the cmgc group, its relative importance in the p. falciparum kinome, and our long-standing interest in the control of cell proliferation and differentiation in the parasite, prompted us to produce a three-species comparative tree of this group .
tkl group
five malarial enzymes appear in the vicinity of the tyrk-like group, including two  that display maximal homology to mapkkk-related or mlk  enzymes upon blastp analysis.
pfb0520w clusters with the tgfβ receptor .
the malarial sequence is much more similar to tgfβ receptors than to mammalian raf, and furthermore, in common with tgfβ receptors, the malarial enzyme has a predicted transmembrane sequence n-terminal to the kinase domain.
mammalian tgfβ receptors assemble as heterodimers, and it remains to be seen whether the malarial enzyme forms a homodimer or has the capacity to coassemble with a mammalian subunit.
absence of members of the ste and tyrk groups
no malarial protein kinase clusters with the ste7/11/ <dig> group, which is consistent with the lack of success of earlier in vitro and in silico attempts at identifying mapkk malarial homologues  <cit>  and points to a divergent organisation of the mapk pathways in malaria parasites .
it is relevant to mention here that one of the p. falciparum nima-related enzymes  possesses an activation site that closely mimics that of mek1/ <dig>  this enzyme, pfnek- <dig> , is able to specifically phosphorylate pfmap- <dig>  in vitro, and to act in synergy with pfmap- <dig> towards the phosphorylation of exogenous substrates  <cit> .
this suggests that pfmap- <dig> activity may be regulated by pfnek- <dig>  however, the physiological relevance of these finding remains to be demonstrated.
our tree indicates that members of the tyrk family are absent, as is the case in yeast and most  unicellular eukaryotes  <cit> .
other clusters and "orphan" kinases
five plasmodium genes form a cluster of nima-related sequences that includes the nima-related kinase nek <dig>  of these five, four are recognised by blastp analysis as being related to the nima/nek family  <cit> , including the well characterised pfnek- <dig> enzyme  <cit> .
the fifth enzyme, mal6p <dig> , does not cluster with the nima-like kinases in other analyses .
several protein kinases appear not to cluster clearly with any defined group, or to constitute small "satellite" clusters.
examples of such "orphan" kinases are  the cluster formed by pfkin , an enzyme previously described as related to the snf <dig> family  <cit> , with two uncharacterised pfpks .
this cluster is located at the base of the camk and agc branches, and does not strongly associate with any established epk group ).
a group of three malarial enzymes, including pfpk <dig> , a previously characterised hri kinase homologue  <cit> , that are similar to mammalian elongation factor kinases, and form a distinct cluster associated to the nima group.
several sequences that are isolated at the base of major branches of the tree, indicating an absence of relatedness to established epk groups.
these include the "p. falciparum exported protein kinase"   <cit> , which forms an isolated branch at the base of the part of the tree containing the cmgc, camk and agc groups, pfl2280w, which is in a similar situation, and a group of two sequences at forming a sister cluster to the branch containing the agc and camk groups.
one of these two sequences, pfpk <dig> , displays relatedness to agc and ste kinases in blast analysis .
so far, four pfepks have been described as appearing as "composite" enzymes displaying features from more than one established epk family.
as mentioned above, pfpk <dig>  and pfcrk- <dig>  both display relatedness to cdks and mapks, and this is confirmed by their position on the tree.
the mapkk-like activation site of pfnek- <dig>  has been discussed above.
the fourth example is that of pfpk <dig> , an enzyme whose c-terminal region carries a sequence which is conserved in mapkks but whose n-terminal region is more closely related to that of pkas  <cit> .
this sequence does not cluster with any well-defined group in the tree, although it associates with uncharacterized pfi1415w in a sister cluster to the major branch containing the agc and camk groups.
whether such "dual" enzymes represent common ancestors to subsequently divergent families which have been conserved in the evolution of the apicomplexan lineage, or whether they arose from domain shuffling between existing kinase genes, remains to be elucidated.
it is possible that additional such "composite" enzymes will be identified, particularly among the pfpks which do not associate with well defined pk groups.
a three-species comparison of cmgc kinases
because of the large number of cmgc-group kinases found in the p. falciparum genome, we carried out a more thorough analysis in which the  <dig> malarial kinases belonging to this group were compared with comprehensive sets of related kinases from the yeast and human genomes .
the phylogenetic tree was constructed in a similar way to that in figure  <dig>   <dig> amino acid positions from the alignment were used in the construction of the tree.
evidence for absence of typical 3-component mapk pathways
in this analysis, both p. falciparum kinases  previously reported as belonging to the erk family clustered, as expected, with the map kinases.
however, in contrast to previous suggestions brought forward before the full complement of mammalian erks had been characterised  <cit> , they do not specifically cluster with erk1/ <dig>  rather, they lie outside the cluster of typical map kinases comprising the p <dig>  jnk and erk1/ <dig> classes from human and yeast.
pfmap- <dig> lies at a basal position relative to the mapk family, indicating no preferential relatedness to any of its subfamilies.
pfmap- <dig>  in contrast, clearly associates with erk <dig>  a recently described member of the erk family which, like pfmap- <dig>  has a large extension at the c-terminus  <cit> .
in the orthologous rat enzyme erk <dig>  a similar extension has been shown to be involved in regulation of enzymatic activity  <cit> .
it has hence been proposed that erk8/ <dig> may not be part of typical three-component  modules which are the hallmark of the erk1/ <dig>  p <dig> and jnk pathways.
formal demonstration that erk8/ <dig> is not regulated by classical meks in mammalian cells is difficult because of the numerous mek homologues present in the genome.
the situation in p. falciparum therefore provides a first clear example that in vivo regulation of a kinase related to erk8/ <dig> does not require a typical mek, since no member of the latter family is present in the parasite's genome .
it is perhaps unsurprising that p. falciparum lacks map kinases of the erk1/erk <dig>  p <dig> of jnk subfamilies, given the absence of mapkks and ste-like mapkkks in the genome.
in summary, our data indicate that although the malaria parasite uses mapk homologues, these are not part of three-component modules – to our knowledge, p. falciparum is the first eukaryote demonstrated to lack such modules.
cell cycle control kinases
three p. falciparum kinases cluster with the cell division kinase group that includes the human cell cycle cdks.
pfpk <dig>  appears orthologous to yeast cdc <dig> and to human cdk1- <dig>  pfpk <dig> displays similar levels  of overall homology to both mammalian cdk <dig> and cdk5; however, in our analysis this enzyme clearly clusters with the former, lending support to the idea that this enzyme is a functional homologue of the major cell cycle control kinases, as previously suggested  <cit> .
the other two malarial enzymes that clearly cluster within the cdk group, pfcrk- <dig>  and pfcrk- <dig> , cannot be assigned an orthology with any yeast kinase.
however, pfcrk- <dig> appears to be related to human cdks such as cdk <dig> and cdk <dig> that are involved in transcriptional control, consistent with earlier reports  <cit>  that this enzyme shares primary structure features with the human pitslre  kinases.
pfmrk  was initially described  <cit>  as a putative homologue of the cdk-activating kinases  such as mammalian cdk <dig>  and subsequently shown to be able to undergo some activation by human cyclin h  and by pfcyc- <dig>  a p. falciparum protein with maximal homology to cyclin h  <cit> .
however, in our tree pfmrk appears not to be included in the cdk <dig> cluster, but instead lies at an intermediate position between the mapk and the cdk groups.
it is relevant to mention here that sequence-based prediction of kinase-cyclin pairs is difficult: for example, pfpk <dig>  a clear cdk1- <dig> orthologue, is unexpectedly activated very efficiently in vitro by human cyclin h  and p <dig> , among other cyclin-related proteins  <cit> .
this may be explained by structural properties making this enzyme very prone to adopt the active conformation  <cit> .
extreme caution must therefore be exercised in predicting precise functions for the four cyclin-related proteins which have been identified so far  <cit> .
the positions of the clusters containing  pfpk <dig>   <cit>  and pfcrk- <dig> , and  pfcrk- <dig> and uncharacterized mal13p <dig> , are consistent with the data in the general tree, and confirm the previously detected relatedness of two of these enzymes to both cdks and mapks.
overall, the number of clear orthologues of cell division kinases in the p. falciparum genome is smaller than that in the yeast or human genomes, and may represent a minimum complement of such kinases that are necessary for the completion of a eukaryotic cell cycle.
alternatively, some cell cycle control functions assured by cdks in human cells may be taken over, in plasmodium, by some of the cmgc kinases with no clear relatedness to established families.
other cmgc kinases
a number of cmgc group kinases interact with factors involved in mrna splicing.
pf11_ <dig> clearly is an orthologue of human prp <dig>  a kinase that is associated with mrna splicing and histone deacetylation and that is conserved in most eukaryotic genomes   <cit> .
human srpk <dig> phosphorylates the "serine-arginine-rich pre-mrna splicing factors" called sr proteins, and homologues are conserved in all eukaryotic genomes  <cit> .
two p. falciparum kinases  cluster with sprk.
both these kinases have an insertion between domains vib and vii that is a distinctive feature of srpks.
previously described pflammer   <cit>  associates with yeast kns <dig>  <cit>  and the related human lammer kinases clk1- <dig> that also phosphorylate sr proteins  <cit> .
other kinases clustering within the cmgc group include a single orthologue of casein kinase 2α .
other eukaryotes have at least  <dig> alpha subunit-encoding genes, emphasizing the relative simplicity of the p. falciparum kinome.
as detected on the general tree , three malarial enzyme cluster with the gsk <dig> family, the most closely related to human gsk3α/β being the recently characterised pfgsk <dig> , which appears to be exported into the host erythrocyte  <cit> .
in several instances our phylogenetic classification of individual kinases differs from the previously reported classification based on blast searches.
there are at least two reasons for this discrepancy.
firstly, our analysis is based on a comprehensive catalogue of protein kinases from p. falciparum, and we have access to comprehensive catalogues from several other organisms.
in contrast, several malarial epks were classified at the time of their initial identification several years ago, when the sequences could be compared only to non-comprehensive sets.
as an example, both p. falciparum mapks were identified before the mammalian erk8/ <dig> enzymes were discovered, and the closest sequences available at the time were those of the erk1/ <dig> family.
secondly, it has been reported that blast performs poorly in assigning orthology between human and c. elegans genes  <cit> .
this is because of extensive independent gene duplication on the lineages leading to the two organisms.
humans and p. falciparum are much more distantly related and there has been extensive gene duplication on the human side.
our data support the view that reliable assignments of orthology between genes in distantly related species might only be assigned through the construction of phylogenetic trees and suggest that comparisons based on blast must be interpreted cautiously.
fikk, a novel, apicomplexa-specific group of epk-related proteins
that only  <dig> typical epks were identified in this search is somewhat surprising, as saccharomyces cerevisiae, whose genome  is half the size of the p. falciparum genome , encodes approximately twice as many enzymes of this family.
in preliminary analyses,  <dig> sequences identified in the hmm search appeared to form a tight cluster that is relatively distantly related to the more typical epk groups discussed above.
based on an amino acid motif corresponding to subdomain ii of the epk catalytic domain, and which is well conserved in members of this novel family, we called this group "fikk".
in addition to the epk catalytic domain-like region, the fikk sequences all have a highly variable n-terminal extension, and in some cases the catalytic domain itself is interrupted by large insertions .
an alignment of the fikk kinase-like domain with the  <dig> typical epks in the p. falciparum genome showed that they share most of the residues that are conserved in the epk catalytic domain.
indeed, with the exception of the glycine triad in subdomain i, all residues which are crucial for phosphotransfer or structural stability of protein kinases, and therefore well conserved throughout the family, are present in all members of this family .
in contrast, no fikk sequence possesses a full glycine triad  in subdomain i. this triad is present in a majority of epks and is involved in positioning the atp molecule in the catalytic cleft  <cit> .
however, one, and sometimes two glycine residues are present in subdomain i of the fikk sequences.
this is also the case in a number of enzymes with demonstrated protein kinase activity from many organisms   <cit> , and it is clearly established that atp binding and phosphotransfer ability is not dependent on the presence of a glycine triad.
although lacking the glycine triad, all fikk sequences possess an n-terminal extension, with a conserved tryptophan residue in the region that corresponds to subdomain i. one of the fikk sequences is represented in plasmodb as two contiguous orfs  separated by a gap.
this is presumably due to erroneous prediction: alignment with other fikks clearly shows these sequences represent two parts of a single member of the fikk family rather than two separate genes.
furthermore, rt-pcr across the two predicted orfs demonstrates that both sections are present on the same mrna molecule.
interestingly, sequencing of the rt-pcr product showed that the open reading frame in the cdna is interrupted by an in-frame stop codon, which is presumably the cause of the misprediction of the gene structure.
that this sequence is cdna than genomic is ascertained by the presence of an intron near the 3'end .
whether pf14_0733/ <dig> is a transcribed pseudogene, or whether a protein can be produced by readthrough of the internal stop codon as has been documented for another p. falciparum gene  <cit> , remains to be determined.
in any case, it appears there are only  <dig> fikk sequences in the genome, instead of the  <dig> that were counted originally .
n pf14_0408
n pfa0380a
y pf11_0060
y pf11_0220
n pf11_0377
mal7p1_73
mal7p1_91
pf11_0060
pf14_0408
pfa0380w
pfi1415w
note: a. this residue corresponds to the first k in the fikk motif.
in addition to the residues conserved in typical epks, several amino-acid motifs are fully conserved in all members of the fikk family .
these can be used to define signature motifs, which allowed us to perform a number of motif searches in various databases, to determine whether members of this epk-like family are present in other organisms.
interestingly, sequences containing such motifs could be retrieved only from apicomplexan species:  <dig> sequences in the p. falciparum genome, one in p. berghei , one in p. yoelii , one in p. knowlesi  and one in p. vivax .
in contrast, no fikk family member was found in the  genomes of p. chabaudi or p. reichenowi.
searches of the nrprot database, which contained sequences representing all eukaryotic and prokaryotic phyla, yielded only the plasmodium sequences mentioned above .
in agreement with the motif searches, blast analysis of the nrprot database with pf10_ <dig> finds  <dig> plasmodium falciparum and one yoelii sequences among the top hits .
weaker hits  are mostly mapks from a variety of organisms.
further investigations using apicomplexan genome project databases  allowed us to identify one such sequence in toxoplasma gondii and one in cryptosporidium parvum.
taken together, these data strongly suggest that the fikk group is specific to apicomplexa, and has undergone a dramatic expansion in p. falciparum.
interestingly, of the  <dig> fikk sequences in the p. falciparum genome,  <dig> are located on chromosome  <dig>  where they are arranged in a contiguous subtelomeric tandem array, a common location for genes involved in antigenic variation such as those of the var/pfemp <dig>  <cit>  or rifin  <cit>  families .
on the tree depicted in fig.
<dig>  these sequences  tend to cluster together.
the structure of the tree  suggests a rapid and presumably recent expansion of the family.
this hypothesis is supported by the presence of the tandem array, an indicator of gene duplication.
furthermore, the presence of only one fikk gene in several other apicomplexan species is consistent with the expansion in p. falciparum being a recent event.
obviously, a definite conclusion about the species distribution of this gene family will have to await the completion of additional genome sequencing projects, especially with respect to other plasmodium species and other apicomplexan genera.
although no experimental evidence is available that associates pk activity with any of the fikk sequences, the fact that all residues required for phosphotransfer and epk folding are present strongly suggests that these proteins are indeed protein kinases.
some fikks have a predicted signal peptide  and/or transmembrane helix  at the n-terminus.
otherwise, aside from their similarity to the kinase domain, no recognised pfam domains are found in these proteins.
two of the fikk sequences have been identified as p. falciparum antigens in the context of immunological studies: the r <dig> trophozoite antigen   <cit>  and the  <dig>  protein .
no function has previously been attributed to either of these proteins.
r <dig> has a large insertion of  <dig> residues, comprising mostly his, lys, asn, ser and asp residues, relative to the other members of the fikk family.
the belonging of r <dig> to a 20-sequence family in the p. falciparum genome has been discovered independently in the context of research into the r <dig> antigen .
features of gene structure
like in many other plasmodial proteins, large extensions rich in charged and/or polar residues, and in some cases repeated amino acid motifs, are found adjacent to the catalytic domain of several pfpks.
several enzymes also carry such sequences as insertions within the catalytic domain.
the function of these elements is as yet undetermined, although there is evidence in some cases  that extensions are absent from the enzymes in parasite protein extracts, presumably as a result from proteolytic cleavage.
in some sequences , large insertions have been mapped to the hinge region between adjacent β-sheets in the n-terminal lobe; hence, it can be argued that such insertions may not interfere with proper folding of the catalytic domain .
organelle targeting
malaria parasites possess two organelles with extra-chromosomal dna: the apicoplast and the mitochondrion.
the apicoplast is a four-membrane organelle carrying a circular  <dig> kb dna whose structure is very similar to that of plastid genomes.
it is specific to the apicomplexa , and thought to originate from secondary endosymbiosis  <cit> .
as is the case for chloroplasts in plants  <cit> , it appears that many genes whose products are essential for apicoplast function and survival have been transferred to the "host cell" nucleus; products of these genes must be addressed back to the organelle.
a bipartite peptide has been identified and shown to be necessary and sufficient for targeting a protein to the apicoplast  <cit> .
the  <dig> kb genome of the apicoplast does not encode any pk, but it is to be expected that protein phosphorylation is necessary for function and maintenance of the organelle.
we used an algorithm available on plasmodb to determine that 5/ <dig> typical epfpks  and 6/ <dig> fikks are predicted to be addressed to the apicoplast .
likewise, four kinases  possess a potential mitochondrion-targeting signal sequence, as defined by the algorithm available on plasmodb  <cit> .
it is important to emphasise that presence or absence of targeting signals relies on gene structure prediction algorithms, which have been demonstrated to be erroneous in some instances ; therefore this must be considered with caution until the 5'end of the cdnas has been sequenced, and targeting to the organelle has been verified experimentally by the transfection of constructs expressing gfp-tagged proteins.
regulatory subunits
proteins devoid of kinase activity but which are known to associate with, and regulate the activity of, epks have been identified in plasmodb.
these include four previously characterised cyclins  which have been demonstrated to associate with histone h <dig> kinase activities in parasite extracts  <cit> , a pka regulatory subunit , which as expected is able to down-regulate pka in parasite extracts , and two putative ck <dig> regulatory subunits .
genes encoding apks
blastp searches of plasmodb were performed using atypical protein kinases  from homo sapiens as queries.
genedb was also used to look for relevant pfam domains .
two members of the rio kinase family were found: pfl1490w  and pfd0975w .
enzymes of this family are involved in rrna processing in s. cerevisiae  <cit> .
we also identified two putative members of the abc <dig> family of atypical protein kinases .
some p. falciparum genes  display regions with low-level similarity to the histidine kinase domain , but the significance of this observation remains to be established.
no significant hits were obtained with a <dig> kinases, alpha kinase, pyruvate dehydrogenase kinase, aminoglycoside phosphotransferases or dna-dependent kinases.
in contrast, we identified a malarial phosphatidyl-inositol- <dig> kinase homologue , in agreement with experimental studies  <cit>  and the presence of a pkb homologue  demonstrating the presence of a phosphatidyl-inositol pathway in the parasite.
however, the pi3k homologue, like two other sequences  related to pi4k, appears not to contain the fat and fatc domains which are present in piks from other organism and have been associated with protein kinase activity  <cit> .
hence, it may be that these three enzymes function solely as phosphatidylinositol kinases, a proposition that requires experimental testing.
overall, these results on malarial apks contrast with those obtained from the recently-sequenced l. major, t. brucei and t. cruzi genomes, where abc <dig> and rio kinases were found, as were pikk , pdhk and alpha kinase family members .
expression pattern of pfpks during the p. falciparum life cycle
data from two studies  <cit>  of the p. falciparum transcriptome during development are available on the plasmodb database.
we compiled these data to obtain a general picture of pfepk gene expression during erythrocytic development .
it is clear that the steady-state level of mrna is developmentally regulated for all the pfpk genes, in accordance with the unique gene expression pattern described in this organism by bozdech et al.
<cit> .
most of the pfepks are expressed in trophozoites and schizonts, but some pk mrnas are clearly predominantly detected in rings, the younger form following erythrocyte invasion.
data from le roch et al.
<cit>  included a transcriptome analysis of additional development stages: free merozoites, gametocytes and sporozoites.
compilation of data from this study indicated that a small number of pfepks are specific to gametocytes, including two of the nima-related kinases , one of the mapks , and pfkin, an enzyme previously described as related to the snf <dig> family .
gametocyte-specific expression had been described in the literature for the latter two enzymes  <cit> .
overall, and despite some discrepancies, there is good agreement between the two studies with respect to pfepk genes, as illustrated by the observation that pfepks whose expression is detected in late schizonts and segmenters by bozdech et al.
are also detected in free merozoites by le roch et al.
at least some of these enzymes are likely to play a role in invasion of the erythrocyte by the merozoite.
as expected, the pk genes that are gametocyte-specific according to le roch et al.
give low intensity signals in the dataset from bozdech et al.
.
CONCLUSIONS
this study has allowed us to classify the  <dig> typical epks encoded by the p. falciparum genome, and to establish the presence of a novel group of epk-related genes, the fikk family, which, from analysis of currently available databases, appears to be specific to apicomplexa and considerably extended in p. falciparum.
the number of genes encoding protein kinases is somewhat smaller than expected from analogy with other organisms.
we cannot exclude that our study, which is based on sequence similarity with epks, may have missed genes encoding proteins with protein kinase activity, but with a primary structure that would be too divergent from that of known epks to be identified.
nevertheless, it is hoped that the present study will facilitate investigations into the regulation of many pathways and processes operating during growth and development of the parasite.
in addition to the fikks, several malarial epks belong to "orphan" groups, as they do not cluster clearly with established epk groups as defined in model organisms.
furthermore, our analysis provides evidence that elements which are usually found in eukaryotes are absent or dramatically modified in malaria parasites.
such elements include mapk pathway components and pkc, for example.
these important divergences between the malarial and human kinomes reflects the vast phylogenetic distance between apicomplexans and opisthokonta, and strengthen our expectations that specific interference with essential functions of the parasite can be achieved through the use of protein kinase inhibitors.

 modulation of protein phosphorylation through the antagonistic effects of protein kinases and protein phosphatases is a major regulatory mechanism of most cellular processes. dysregulation of protein phosphorylation in human cells plays a major role in many diseases such as cancers and neurodegenerative disorders. this has prompted the search for drugs targeting protein kinases, an endeavour which led in to the commercialisation of gleevec, the first protein kinase inhibitor used as a drug for human disease. additional molecules targeting protein kinases are in clinical trial, and significant developments in this field are expected in the next few years. some of the most devastating infectious diseases are caused by protists such as malaria parasites and trypanosomatids: hence, about half the global population lives in malarious areas, with 10% of the world population contracting the disease each year, which results in 1– million annual deaths. the essential role played by eukaryotic protein kinases in crucial cellular functions makes them attractive potential targets for drugs against such eukaryotic infectious agents. malaria parasites have a complex life cycle. infection of human beings by plasmodium falciparum, the species responsible for the lethal form of human malaria, begins with the bite of an infected anopheles mosquito, which delivers sporozoites into the bloodstream. these cells establish an infection inside hepatocytes, where they undergo an intense multiplication generating several thousand merozoites, a process called exo-erythrocytic schizogony. the merozoites invade erythrocytes, where they also undergo schizogony, the process that is responsible for malaria pathogenesis. some merozoites, however, arrest the cell cycle and differentiate into male or female gametocytes, which are infective to the mosquito. once ingested by the insect, the gametocytes develop into gametes and fuse into a zygote. further development in the mosquito involves a process of sporogony, producing sporozoites that accumulate in the salivary glands and are now ready to infect a new human host. the observation that many parasitic epks display profound structural and functional divergences from their counterparts in their vertebrate hosts suggests that specific inhibition is an attainable goal. the availability of plasmodb, a genomic database for plasmodium falciparum, now permits a systematic analysis of the entire complement of epks encoded in the genome of this pathogen, an important milestone both in our understanding of plasmodium biology and in the definition of potential novel drug targets. prior to the genomic era, the initial classification system of hanks and quinn distributed epks into four major groups: • the cyclic-nucleotide- and calcium/phospholipid-dependent kinases; • the cmgc group, constituted of the cyclin-dependent-, mitogen-activated-, glycogen-synthase- and cdk-like kinases; • the calmodulin-dependent kinases, and • the tyrosine kinases. epks that did not clearly fit into any of these groups were placed into the opk group. the primary structure of all enzymes in these groups conform to the model described by hanks, in which the catalytic domain is subdivided into eleven subdomains, which can be aligned across all groups. in addition to the "typical" epks, several enzymes possessing protein kinase activity, but which are unrelated to epks at the primary structure level, have been identified and termed "atypical protein kinases". these include phosphatidyl-inositol 3' kinase, dna-dependent protein kinase, and members of pyruvate dehydrogenase kinase family. exhaustive analyses of the kinome of some model organisms have now been published. the kinome of s. cerevisiae contains epks, and the genomes of d. melanogaster, c. elegans and h. sapiens comprise and 510– epk-coding genes, respectively. on the basis of this wealth of new data, three additional major epk groups were recognized group; • the ste group, which includes many enzymes functioning in mapk pathways, although the mapks themselves belong to the cmgc group; • the tyrosine kinase-like group, which, as its name indicates, includes enzymes that are related to those in the tyrk group, although they are serine-threonine protein kinases. furthermore, a description of the non-receptor serine/threonine protein kinases of the plant arabidopsis thaliana has recently been published. comparative examination of this and previously available kinomes has demonstrated that members of all major epk groups can be found in yeast, worms, insects, mammals and plants, with the exception of tyrks, which are not found in yeast. that most tyrks function in hormone-response receptor-linked pathways suggests that this family arose as an adaptation to the needs for intercellular communication in multicellular organisms. it has however been reported recently that a few unicellular eukaryotes possess putative tyrk family members. despite the fact that most serine-threonine epks groups are found in all eukaryotes, indicating that their appearance occurred early in evolution, each of the kinomes has nevertheless its specificities. a striking feature in this respect is the considerable extension of some epk families in some organisms but not in others. for example, yeast and drosophila have and members of the casein kinase group respectively, whereas the c. elegans genome encodes ck1-related genes. with the exception of the plant a. thaliana, all eukaryotes whose kinome has been characterised, from yeast to man, belong to the opisthokonta phylogenetic group. as depicted in fig. this lineage represents only one small branch of the eukaryotic tree. several eukaryotes of high medical importance, such as malaria parasites or trypanosomes, belong to phylogenetic groups that are vastly distant from both the opisthokonta and planta branches. this is reflected by many profound peculiarities in their basic biology. divergences from model eukaryotes can also be expected not only at the level of individual protein kinases of these organisms, but at the level of their kinome as well. as is documented below, our analysis of the p. falciparum kinome confirms this prediction.  overview of the tree sequences related to epks were retrieved from plasmodb and used to construct a phylogenetic tree as described in the methods section. the tree of the p. falciparum kinome indicates that although the parasite possesses enzymes belonging to most of the major serine/threonine kinase groups, as described in the following paragraphs, several enzymes do not cluster with any of these groups. ck group only one malarial kinase, the previously described pfck, clearly falls within this group, which is vastly expanded in some other kinomes. agc group five malarial kinases cluster within this group, three of which have been characterized: the camp-dependent pfpka, the cgmp-dependent pfpkg, and pfpkb, an enzyme displaying maximal similarity to akt/pkb. in other eukaryotes, pkb functions in the pi3k-dependent pathway; a pi3k kinase homologue is present in the p. falciparum genome. two additional sequences form a separate cluster attached to the base of the agc branch. there appears to be no clear member of the pkc subfamily. camk group the main branch of the tree that contains the human camks also contains pfepks, which underlines the importance of calcium signalling in the parasite. a tight cluster is formed by five of these enzymes, which share the overall structure of the calcium-dependent protein kinases found in plants and ciliates but not in metazoans. cdpks are characterised by the presence of a kinase catalytic domain located on the same polypeptide as four ef-hand calcium-binding domains. four of these enzymes have been described previously: pfcdpk, pfcdpk, pfcdkp and more recently pfcdpk. the latter enzyme is expressed in sexual stages and was shown to be essential for development of the parasite in the mosquito, through mediating cell cycle resumption during male gametocyte exflagellation. a fifth cdpk, which like the four cited above possesses four ef-hand motifs, has been discovered in the present study. pf11_ appears to be related to cdpks, but contains only one ef-hand motif. pfpk constitutes a sister branch to the cdpk group. this enzyme was previously characterized as being related to the camk family, and has no ef-hand domain. no malarial kinase clusters closely with the mammalian camks used to anchor the tree. six other sequences, however, form a sister branch to the cluster that contains the cdpks; only one of these six sequences possesses an ef-hand domain. the camk activity described as crucial for ookinete development in the mosquito vector is likely to be associated with one of the enzymes in this group. cmgc group eighteen malarial kinases cluster within this group, which makes it the most prominent group in the plasmodium kinome. interestingly, in other eukaryotic systems a majority of cmgc kinases are involved in the control of cell proliferation and development, and their relative abundance in the p. falciparum kinome may reflect the variety of successive proliferative and non-proliferative stages which constitute the life cycle of malaria parasites. six enzymes are related to the cyclin-dependent kinase family, of which were identified previously, the last one having been discovered during the present analysis. two previously characterised mitogen-activated protein kinases, pfmap- and pfmap-, cluster together with a member of the mapk family, as expected. two enzymes, pfpk and pfcrk-, display features of both cdks and mapks according to blastp analysis. their position either in a cluster that is intermediate between the cdk and the mapk groups, or in a cluster at the base of the cdk/mapk/gsk branch, is consistent with these early observations. three gsk3-related kinases, two of which have been characterised previously, form a cluster within the cmgc group. four additional enzymes form another cluster that includes human clk one of which is a previously described lammer-related kinase. the complexity of the cmgc group, its relative importance in the p. falciparum kinome, and our long-standing interest in the control of cell proliferation and differentiation in the parasite, prompted us to produce a three-species comparative tree of this group. tkl group five malarial enzymes appear in the vicinity of the tyrk-like group, including two that display maximal homology to mapkkk-related or mlk enzymes upon blastp analysis. pfb0520w clusters with the tgfβ receptor. the malarial sequence is much more similar to tgfβ receptors than to mammalian raf, and furthermore, in common with tgfβ receptors, the malarial enzyme has a predicted transmembrane sequence n-terminal to the kinase domain. mammalian tgfβ receptors assemble as heterodimers, and it remains to be seen whether the malarial enzyme forms a homodimer or has the capacity to coassemble with a mammalian subunit. absence of members of the ste and tyrk groups no malarial protein kinase clusters with the ste7/11/ group, which is consistent with the lack of success of earlier in vitro and in silico attempts at identifying mapkk malarial homologues and points to a divergent organisation of the mapk pathways in malaria parasites. it is relevant to mention here that one of the p. falciparum nima-related enzymes possesses an activation site that closely mimics that of mek1/ this enzyme, pfnek-, is able to specifically phosphorylate pfmap- in vitro, and to act in synergy with pfmap- towards the phosphorylation of exogenous substrates. this suggests that pfmap- activity may be regulated by pfnek- however, the physiological relevance of these finding remains to be demonstrated. our tree indicates that members of the tyrk family are absent, as is the case in yeast and most unicellular eukaryotes. other clusters and "orphan" kinases five plasmodium genes form a cluster of nima-related sequences that includes the nima-related kinase nek of these five, four are recognised by blastp analysis as being related to the nima/nek family, including the well characterised pfnek- enzyme. the fifth enzyme, mal6p, does not cluster with the nima-like kinases in other analyses. several protein kinases appear not to cluster clearly with any defined group, or to constitute small "satellite" clusters. examples of such "orphan" kinases are the cluster formed by pfkin, an enzyme previously described as related to the snf family, with two uncharacterised pfpks. this cluster is located at the base of the camk and agc branches, and does not strongly associate with any established epk group ). a group of three malarial enzymes, including pfpk, a previously characterised hri kinase homologue, that are similar to mammalian elongation factor kinases, and form a distinct cluster associated to the nima group. several sequences that are isolated at the base of major branches of the tree, indicating an absence of relatedness to established epk groups. these include the "p. falciparum exported protein kinase", which forms an isolated branch at the base of the part of the tree containing the cmgc, camk and agc groups, pfl2280w, which is in a similar situation, and a group of two sequences at forming a sister cluster to the branch containing the agc and camk groups. one of these two sequences, pfpk, displays relatedness to agc and ste kinases in blast analysis. so far, four pfepks have been described as appearing as "composite" enzymes displaying features from more than one established epk family. as mentioned above, pfpk and pfcrk- both display relatedness to cdks and mapks, and this is confirmed by their position on the tree. the mapkk-like activation site of pfnek- has been discussed above. the fourth example is that of pfpk, an enzyme whose c-terminal region carries a sequence which is conserved in mapkks but whose n-terminal region is more closely related to that of pkas. this sequence does not cluster with any well-defined group in the tree, although it associates with uncharacterized pfi1415w in a sister cluster to the major branch containing the agc and camk groups. whether such "dual" enzymes represent common ancestors to subsequently divergent families which have been conserved in the evolution of the apicomplexan lineage, or whether they arose from domain shuffling between existing kinase genes, remains to be elucidated. it is possible that additional such "composite" enzymes will be identified, particularly among the pfpks which do not associate with well defined pk groups. a three-species comparison of cmgc kinases because of the large number of cmgc-group kinases found in the p. falciparum genome, we carried out a more thorough analysis in which the malarial kinases belonging to this group were compared with comprehensive sets of related kinases from the yeast and human genomes. the phylogenetic tree was constructed in a similar way to that in figure amino acid positions from the alignment were used in the construction of the tree. evidence for absence of typical 3-component mapk pathways in this analysis, both p. falciparum kinases previously reported as belonging to the erk family clustered, as expected, with the map kinases. however, in contrast to previous suggestions brought forward before the full complement of mammalian erks had been characterised, they do not specifically cluster with erk1/ rather, they lie outside the cluster of typical map kinases comprising the p jnk and erk1/ classes from human and yeast. pfmap- lies at a basal position relative to the mapk family, indicating no preferential relatedness to any of its subfamilies. pfmap- in contrast, clearly associates with erk a recently described member of the erk family which, like pfmap- has a large extension at the c-terminus. in the orthologous rat enzyme erk a similar extension has been shown to be involved in regulation of enzymatic activity. it has hence been proposed that erk8/ may not be part of typical three-component modules which are the hallmark of the erk1/ p and jnk pathways. formal demonstration that erk8/ is not regulated by classical meks in mammalian cells is difficult because of the numerous mek homologues present in the genome. the situation in p. falciparum therefore provides a first clear example that in vivo regulation of a kinase related to erk8/ does not require a typical mek, since no member of the latter family is present in the parasite's genome. it is perhaps unsurprising that p. falciparum lacks map kinases of the erk1/erk p of jnk subfamilies, given the absence of mapkks and ste-like mapkkks in the genome. in summary, our data indicate that although the malaria parasite uses mapk homologues, these are not part of three-component modules – to our knowledge, p. falciparum is the first eukaryote demonstrated to lack such modules. cell cycle control kinases three p. falciparum kinases cluster with the cell division kinase group that includes the human cell cycle cdks. pfpk appears orthologous to yeast cdc and to human cdk1- pfpk displays similar levels of overall homology to both mammalian cdk and cdk5; however, in our analysis this enzyme clearly clusters with the former, lending support to the idea that this enzyme is a functional homologue of the major cell cycle control kinases, as previously suggested. the other two malarial enzymes that clearly cluster within the cdk group, pfcrk- and pfcrk-, cannot be assigned an orthology with any yeast kinase. however, pfcrk- appears to be related to human cdks such as cdk and cdk that are involved in transcriptional control, consistent with earlier reports that this enzyme shares primary structure features with the human pitslre kinases. pfmrk was initially described as a putative homologue of the cdk-activating kinases such as mammalian cdk and subsequently shown to be able to undergo some activation by human cyclin h and by pfcyc- a p. falciparum protein with maximal homology to cyclin h. however, in our tree pfmrk appears not to be included in the cdk cluster, but instead lies at an intermediate position between the mapk and the cdk groups. it is relevant to mention here that sequence-based prediction of kinase-cyclin pairs is difficult: for example, pfpk a clear cdk1- orthologue, is unexpectedly activated very efficiently in vitro by human cyclin h and p, among other cyclin-related proteins. this may be explained by structural properties making this enzyme very prone to adopt the active conformation. extreme caution must therefore be exercised in predicting precise functions for the four cyclin-related proteins which have been identified so far. the positions of the clusters containing pfpk and pfcrk-, and pfcrk- and uncharacterized mal13p, are consistent with the data in the general tree, and confirm the previously detected relatedness of two of these enzymes to both cdks and mapks. overall, the number of clear orthologues of cell division kinases in the p. falciparum genome is smaller than that in the yeast or human genomes, and may represent a minimum complement of such kinases that are necessary for the completion of a eukaryotic cell cycle. alternatively, some cell cycle control functions assured by cdks in human cells may be taken over, in plasmodium, by some of the cmgc kinases with no clear relatedness to established families. other cmgc kinases a number of cmgc group kinases interact with factors involved in mrna splicing. pf11_ clearly is an orthologue of human prp a kinase that is associated with mrna splicing and histone deacetylation and that is conserved in most eukaryotic genomes. human srpk phosphorylates the "serine-arginine-rich pre-mrna splicing factors" called sr proteins, and homologues are conserved in all eukaryotic genomes. two p. falciparum kinases cluster with sprk. both these kinases have an insertion between domains vib and vii that is a distinctive feature of srpks. previously described pflammer associates with yeast kns and the related human lammer kinases clk1- that also phosphorylate sr proteins. other kinases clustering within the cmgc group include a single orthologue of casein kinase 2α. other eukaryotes have at least alpha subunit-encoding genes, emphasizing the relative simplicity of the p. falciparum kinome. as detected on the general tree, three malarial enzyme cluster with the gsk family, the most closely related to human gsk3α/β being the recently characterised pfgsk, which appears to be exported into the host erythrocyte. in several instances our phylogenetic classification of individual kinases differs from the previously reported classification based on blast searches. there are at least two reasons for this discrepancy. firstly, our analysis is based on a comprehensive catalogue of protein kinases from p. falciparum, and we have access to comprehensive catalogues from several other organisms. in contrast, several malarial epks were classified at the time of their initial identification several years ago, when the sequences could be compared only to non-comprehensive sets. as an example, both p. falciparum mapks were identified before the mammalian erk8/ enzymes were discovered, and the closest sequences available at the time were those of the erk1/ family. secondly, it has been reported that blast performs poorly in assigning orthology between human and c. elegans genes. this is because of extensive independent gene duplication on the lineages leading to the two organisms. humans and p. falciparum are much more distantly related and there has been extensive gene duplication on the human side. our data support the view that reliable assignments of orthology between genes in distantly related species might only be assigned through the construction of phylogenetic trees and suggest that comparisons based on blast must be interpreted cautiously. fikk, a novel, apicomplexa-specific group of epk-related proteins that only typical epks were identified in this search is somewhat surprising, as saccharomyces cerevisiae, whose genome is half the size of the p. falciparum genome, encodes approximately twice as many enzymes of this family. in preliminary analyses, sequences identified in the hmm search appeared to form a tight cluster that is relatively distantly related to the more typical epk groups discussed above. based on an amino acid motif corresponding to subdomain ii of the epk catalytic domain, and which is well conserved in members of this novel family, we called this group "fikk". in addition to the epk catalytic domain-like region, the fikk sequences all have a highly variable n-terminal extension, and in some cases the catalytic domain itself is interrupted by large insertions. an alignment of the fikk kinase-like domain with the typical epks in the p. falciparum genome showed that they share most of the residues that are conserved in the epk catalytic domain. indeed, with the exception of the glycine triad in subdomain i, all residues which are crucial for phosphotransfer or structural stability of protein kinases, and therefore well conserved throughout the family, are present in all members of this family. in contrast, no fikk sequence possesses a full glycine triad in subdomain i. this triad is present in a majority of epks and is involved in positioning the atp molecule in the catalytic cleft. however, one, and sometimes two glycine residues are present in subdomain i of the fikk sequences. this is also the case in a number of enzymes with demonstrated protein kinase activity from many organisms, and it is clearly established that atp binding and phosphotransfer ability is not dependent on the presence of a glycine triad. although lacking the glycine triad, all fikk sequences possess an n-terminal extension, with a conserved tryptophan residue in the region that corresponds to subdomain i. one of the fikk sequences is represented in plasmodb as two contiguous orfs separated by a gap. this is presumably due to erroneous prediction: alignment with other fikks clearly shows these sequences represent two parts of a single member of the fikk family rather than two separate genes. furthermore, rt-pcr across the two predicted orfs demonstrates that both sections are present on the same mrna molecule. interestingly, sequencing of the rt-pcr product showed that the open reading frame in the cdna is interrupted by an in-frame stop codon, which is presumably the cause of the misprediction of the gene structure. that this sequence is cdna than genomic is ascertained by the presence of an intron near the 3'end. whether pf14_0733/ is a transcribed pseudogene, or whether a protein can be produced by readthrough of the internal stop codon as has been documented for another p. falciparum gene, remains to be determined. in any case, it appears there are only fikk sequences in the genome, instead of the that were counted originally. n pf14_0408 n pfa0380a y pf11_0060 y pf11_0220 n pf11_0377 mal7p1_73 mal7p1_91 pf11_0060 pf14_0408 pfa0380w pfi1415w note: a. this residue corresponds to the first k in the fikk motif. in addition to the residues conserved in typical epks, several amino-acid motifs are fully conserved in all members of the fikk family. these can be used to define signature motifs, which allowed us to perform a number of motif searches in various databases, to determine whether members of this epk-like family are present in other organisms. interestingly, sequences containing such motifs could be retrieved only from apicomplexan species: sequences in the p. falciparum genome, one in p. berghei, one in p. yoelii, one in p. knowlesi and one in p. vivax. in contrast, no fikk family member was found in the genomes of p. chabaudi or p. reichenowi. searches of the nrprot database, which contained sequences representing all eukaryotic and prokaryotic phyla, yielded only the plasmodium sequences mentioned above. in agreement with the motif searches, blast analysis of the nrprot database with pf10_ finds plasmodium falciparum and one yoelii sequences among the top hits. weaker hits are mostly mapks from a variety of organisms. further investigations using apicomplexan genome project databases allowed us to identify one such sequence in toxoplasma gondii and one in cryptosporidium parvum. taken together, these data strongly suggest that the fikk group is specific to apicomplexa, and has undergone a dramatic expansion in p. falciparum. interestingly, of the fikk sequences in the p. falciparum genome, are located on chromosome where they are arranged in a contiguous subtelomeric tandem array, a common location for genes involved in antigenic variation such as those of the var/pfemp or rifin families. on the tree depicted in fig. these sequences tend to cluster together. the structure of the tree suggests a rapid and presumably recent expansion of the family. this hypothesis is supported by the presence of the tandem array, an indicator of gene duplication. furthermore, the presence of only one fikk gene in several other apicomplexan species is consistent with the expansion in p. falciparum being a recent event. obviously, a definite conclusion about the species distribution of this gene family will have to await the completion of additional genome sequencing projects, especially with respect to other plasmodium species and other apicomplexan genera. although no experimental evidence is available that associates pk activity with any of the fikk sequences, the fact that all residues required for phosphotransfer and epk folding are present strongly suggests that these proteins are indeed protein kinases. some fikks have a predicted signal peptide and/or transmembrane helix at the n-terminus. otherwise, aside from their similarity to the kinase domain, no recognised pfam domains are found in these proteins. two of the fikk sequences have been identified as p. falciparum antigens in the context of immunological studies: the r trophozoite antigen and the protein. no function has previously been attributed to either of these proteins. r has a large insertion of residues, comprising mostly his, lys, asn, ser and asp residues, relative to the other members of the fikk family. the belonging of r to a 20-sequence family in the p. falciparum genome has been discovered independently in the context of research into the r antigen. features of gene structure like in many other plasmodial proteins, large extensions rich in charged and/or polar residues, and in some cases repeated amino acid motifs, are found adjacent to the catalytic domain of several pfpks. several enzymes also carry such sequences as insertions within the catalytic domain. the function of these elements is as yet undetermined, although there is evidence in some cases that extensions are absent from the enzymes in parasite protein extracts, presumably as a result from proteolytic cleavage. in some sequences, large insertions have been mapped to the hinge region between adjacent β-sheets in the n-terminal lobe; hence, it can be argued that such insertions may not interfere with proper folding of the catalytic domain. organelle targeting malaria parasites possess two organelles with extra-chromosomal dna: the apicoplast and the mitochondrion. the apicoplast is a four-membrane organelle carrying a circular kb dna whose structure is very similar to that of plastid genomes. it is specific to the apicomplexa, and thought to originate from secondary endosymbiosis. as is the case for chloroplasts in plants, it appears that many genes whose products are essential for apicoplast function and survival have been transferred to the "host cell" nucleus; products of these genes must be addressed back to the organelle. a bipartite peptide has been identified and shown to be necessary and sufficient for targeting a protein to the apicoplast. the kb genome of the apicoplast does not encode any pk, but it is to be expected that protein phosphorylation is necessary for function and maintenance of the organelle. we used an algorithm available on plasmodb to determine that 5/ typical epfpks and 6/ fikks are predicted to be addressed to the apicoplast. likewise, four kinases possess a potential mitochondrion-targeting signal sequence, as defined by the algorithm available on plasmodb. it is important to emphasise that presence or absence of targeting signals relies on gene structure prediction algorithms, which have been demonstrated to be erroneous in some instances; therefore this must be considered with caution until the 5'end of the cdnas has been sequenced, and targeting to the organelle has been verified experimentally by the transfection of constructs expressing gfp-tagged proteins. regulatory subunits proteins devoid of kinase activity but which are known to associate with, and regulate the activity of, epks have been identified in plasmodb. these include four previously characterised cyclins which have been demonstrated to associate with histone h kinase activities in parasite extracts, a pka regulatory subunit, which as expected is able to down-regulate pka in parasite extracts, and two putative ck regulatory subunits. genes encoding apks blastp searches of plasmodb were performed using atypical protein kinases from homo sapiens as queries. genedb was also used to look for relevant pfam domains. two members of the rio kinase family were found: pfl1490w and pfd0975w. enzymes of this family are involved in rrna processing in s. cerevisiae. we also identified two putative members of the abc family of atypical protein kinases. some p. falciparum genes display regions with low-level similarity to the histidine kinase domain, but the significance of this observation remains to be established. no significant hits were obtained with a kinases, alpha kinase, pyruvate dehydrogenase kinase, aminoglycoside phosphotransferases or dna-dependent kinases. in contrast, we identified a malarial phosphatidyl-inositol- kinase homologue, in agreement with experimental studies and the presence of a pkb homologue demonstrating the presence of a phosphatidyl-inositol pathway in the parasite. however, the pi3k homologue, like two other sequences related to pi4k, appears not to contain the fat and fatc domains which are present in piks from other organism and have been associated with protein kinase activity. hence, it may be that these three enzymes function solely as phosphatidylinositol kinases, a proposition that requires experimental testing. overall, these results on malarial apks contrast with those obtained from the recently-sequenced l. major, t. brucei and t. cruzi genomes, where abc and rio kinases were found, as were pikk, pdhk and alpha kinase family members. expression pattern of pfpks during the p. falciparum life cycle data from two studies of the p. falciparum transcriptome during development are available on the plasmodb database. we compiled these data to obtain a general picture of pfepk gene expression during erythrocytic development. it is clear that the steady-state level of mrna is developmentally regulated for all the pfpk genes, in accordance with the unique gene expression pattern described in this organism by bozdech et al.. most of the pfepks are expressed in trophozoites and schizonts, but some pk mrnas are clearly predominantly detected in rings, the younger form following erythrocyte invasion. data from le roch et al. included a transcriptome analysis of additional development stages: free merozoites, gametocytes and sporozoites. compilation of data from this study indicated that a small number of pfepks are specific to gametocytes, including two of the nima-related kinases, one of the mapks, and pfkin, an enzyme previously described as related to the snf family. gametocyte-specific expression had been described in the literature for the latter two enzymes. overall, and despite some discrepancies, there is good agreement between the two studies with respect to pfepk genes, as illustrated by the observation that pfepks whose expression is detected in late schizonts and segmenters by bozdech et al. are also detected in free merozoites by le roch et al. at least some of these enzymes are likely to play a role in invasion of the erythrocyte by the merozoite. as expected, the pk genes that are gametocyte-specific according to le roch et al. give low intensity signals in the dataset from bozdech et al..  this study has allowed us to classify the typical epks encoded by the p. falciparum genome, and to establish the presence of a novel group of epk-related genes, the fikk family, which, from analysis of currently available databases, appears to be specific to apicomplexa and considerably extended in p. falciparum. the number of genes encoding protein kinases is somewhat smaller than expected from analogy with other organisms. we cannot exclude that our study, which is based on sequence similarity with epks, may have missed genes encoding proteins with protein kinase activity, but with a primary structure that would be too divergent from that of known epks to be identified. nevertheless, it is hoped that the present study will facilitate investigations into the regulation of many pathways and processes operating during growth and development of the parasite. in addition to the fikks, several malarial epks belong to "orphan" groups, as they do not cluster clearly with established epk groups as defined in model organisms. furthermore, our analysis provides evidence that elements which are usually found in eukaryotes are absent or dramatically modified in malaria parasites. such elements include mapk pathway components and pkc, for example. these important divergences between the malarial and human kinomes reflects the vast phylogenetic distance between apicomplexans and opisthokonta, and strengthen our expectations that specific interference with essential functions of the parasite can be achieved through the use of protein kinase inhibitors. 
using a variety of bioinformatics tools, we identified 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: that several malarial sequences did not cluster within any of the known epk groups; that the cmgc group, whose members are usually involved in the control of cell proliferation, had the highest number of malarial epks; and that no malarial epk clustered with the tyrosine kinase or ste groups, pointing to the absence of three-component mapk modules in the parasite. the fikk family seems restricted to apicomplexa, with members in p. falciparum and just one member in some other apicomplexan species. a novel family of epk-related sequences was identified and called fikk, on the basis of a conserved amino acid motif. eukaryotic protein kinases form a large family of enzymes with crucial roles in most cellular processes; hence malarial epks represent potential drug targets. 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. the p. falciparum genome has been sequenced, which provides new opportunities for the identification of novel drug targets. we report an exhaustive analysis of the p. falciparum genomic database aimed at identifying and classifying all epks in this organism.
 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 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 aimed at identifying and classifying all epks in this organism.  using a variety of bioinformatics tools, we identified 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: that several malarial sequences did not cluster within any of the known epk groups; that the cmgc group, whose members are usually involved in the control of cell proliferation, had the highest number of malarial epks; and that no malarial epk clustered with the tyrosine kinase or ste groups, pointing to the absence of three-component mapk modules in the parasite. a novel family of 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 members in p. falciparum and just one member in some other apicomplexan species.  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. 