Malaria: An overview of disease biology and drug targets
1.6 The kinome of the malaria parasite
1.6.7 Orphan kinases: The Plasmodium falciparum kinome contains several kinases that do not resemble structurally to any of the established ePK groups, and hence are classified under orphan
kinases. PfPK7 is one of these orphan kinases which have been well studied (Figure 1.10). C- terminal lobe of PfPK7 shows highest homology to MAPKKs, while its N-terminal domain is homologous to fungal PKAs (Dorin, Semblat et al. 2005).Parasites in which PfPK7 is deleted shows slower growth rate, produces lesser number of daughter merozoites, as well as
demonstrate impairment in oocyst formation (Dorin-Semblat, Sicard et al. 2008).Another well studied orphan kinase from Plasmodium is PfPK9 (Philip and Haystead 2007) , which demonstrates the ability to auto-phosphorylate itself as well as phosphorylate exogenous substrates in vitro. Auto-phosphorylation specifically occurs on 3 residues. When any of the critical residues are mutated, enzyme activity is abolished, which suggests a potentially complex mechanism of regulation of this kinase in vivo. Recombinant PfPK9 displays selective phosphorylation of an E2 ubiquitin-conjugating enzyme from parasite extract, thereby down- regulating its ubiquitin-conjugating activity (Philip and Haystead 2007). These findings suggest the pivotal role played by PfPK9 in proteasome regulation of Plasmodium.A distinct cluster of twenty one orphan kinases in the malaria parasite belong to the FIKKs. These kinases appear to be solely present in apicomplexans, with a maximum of one enzyme in most species.
These kinases possess a Plasmodium export element (PEXEL) motif (Schneider and Mercereau- Puijalon 2005), and one of the FIKKs have been shown to localize in the host RBC (Nunes, Goldring et al. 2007).
1.6.8 Atypical Protein Kinases (aPKs): Atypical protein kinases are grouped into a separate class due to their lack of sequence similarity to established ePK domains, but have been demonstrated experimentally to possess protein kinase activity. All aPK families are invariably small; many families have just a single member in vertebrates, and none in lower organisms.
Other aPK families might be discovered in the near future using biochemical methods, but since all of the discovered aPK families are small, it can be understood their distribution in the genome is rare. Atypical protein kinases can be further classified into the following families.
1.6.8.1 Alpha Kinases:The earliest discovered kinase of this family is the heavy chains of myosin kinases from the amoeba Dictyostelium discoideum. Although these kinases are evolutionarily restricted, they are homologous to the commonly found eF2 kinases. Several other kinases from mammalian origin have been shown to be homologous to alpha kinases. The channel kinases Chak1 and Chak2, known to be multi-purpose trans-membrane proteins, which act as kinases as well as ion channels are also found to be homologous to alpha kinases. Study of the 3D structure of CHAK1 gene has (Yamaguchi, Matsushita et al. 2001)revealed a clear structural similarity to the ePK domain. Kinase activity has been demonstrated in several members of this aPK family
20
Figure 1.11: Crystal structure of orphan kinase PfPK7 from Plasmodium falciparum (PDB code: 2PML)(Merckx, Echalier et al. 2008). Structure was re-drawn from the original pdb file using UCSF Chimera 1.10.
1.6.8.2Phosphatidyl inositol 3’ kinase-related kinase family (PIKK):This family of aPK contains either a phosphatidyl inositol 3 or phosphatidyl inositol 4 kinase , flankedby a FAT domain at the N-terminal and a FATC domain at its C-terminal (Bosotti, Isacchi et al. 2000).
Human genome contains 6 members of this family and 5 of them have been shown to have distinct protein kinase activity. Multiple sequence alignment demonstrates that PI34K domains belong to a distinct domain subfamily. The PI34K domain is homologous to ePK domain; with a strong similarity in structures and conservation of critical catalytic residues. This includes the catalytic asp residue, the DFG and the HRD motifs (Walker, Perisic et al. 1999).
1.6.8.3The A6 kinase family:The A6 family consists of the related human A6r and A6 genes, along with its close homologs in Drosophila, C. elegans and yeast. The first report in which an A6 kinase was first cloned and expressed was in 1994. This study demonstrated tyrosine kinase activity of A6 in bacterial expression system (Beeler, LaRochelle et al. 1994). A6r was soon discovered. Both the kinases were found to bind ATP but could not yield kinase activity in vitro(Rohwer, Kittstein et al. 1999).
1.6.8.4 ABC1/ADCK family:This conserved family of aPKs was discovered as putative kinases by computational methods such as Protein-Blast and HMMs. ABC1/ADCK domains are weakly homologous to the ePK domain, but most conserved catalytic motifs are highly conserved (Leonard, Aravind et al. 1998). Although these kinases lack an overall sequence similarity with the ePK domain, yet these kinases contain motifs which are exclusive toprotein kinases. These include the kinase catalytic VAIK motif, the DFG motif, and a QTD motif.
1.6.8.5 Pyruvate Dehydrogenase Kinase family (PDK):This family of kinases is found in the mitochondria and contains a domain homologous to the prokaryotic histidine kinases.
Biochemical experiments have revealed that PDKs phosphorylate serine rather thanhistidine.
Published crystal structures (Machius, Chuang et al. 2001, Steussy, Popov et al. 2001) confirm that the PDK domain is homologous to that of histidine kinases, and is not related to the typical ePK domain.
1.6.8.6Bromodomain Kinase family (BRDs):This family of kinases consists of the BRD2 kinase and its near homologs in human and other model organisms. Dennis and Green (1996) BRD2 was first identified as an auto-phosphorylating protein specifically active in nuclear extracts of Hela cells (Denis and Green 1996). Studies in which recombinant BRD2 was expressed in E.
coli, the recombinant protein showed kinase activity in vitro. Mutation of putative catalytic K578 resulted in abolished kinase activity. In another study recombinant BRD2 purified from COS cells also demonstrated in vitro kinase activity.
1.6.8.7 TAF – TATA binding factor associated factors:TAF1 gene is known to be a component of the basal transcriptional machinery in all eukaryotes. It has no close homologs. It was reported that TAF1 is a protein kinase and contains two lobes which are independent of each other and can phosphorylate another basal transcription factor RAP74 (Dikstein, Ruppert et al. 1996).
Immune-purified TFIID from Hela cells as well as cloned TAF1in insect cells andE. Coli showed in vitro kinase activities despite significant sequence similarity to other protein kinases.
Later studies have confirmed similar findings (O'Brien and Tjian 1998, Solow, Salunek et al.
2001). It was revealed that TAF1L, a retro-transposed copy of TAF1is present in human and other primates. This kinase was expressed during spermatogenesis and is a substitute for TAF1 (Wang and Page 2002).
22
1.6.8.8 BCR:This gene isbest known as the fusion partner of Abl kinase in leukemia. TheBCR gene also shows auto- and trans-phosphorylation activities due to conserved cysteines and tyrosines(Maru and Witte 1991). The human isoform as well as the Drosophila counterpartis
~70% and ~ 36% identical to ePK respectively but both lack the N-terminal putative kinase domain.
1.6.8.9 FASTK:The human Fas-activated kinase was characterized by as a novel kinase that was de-phosphorylated and activated during apoptosis (Tian, Taupin et al. 1995). Differential expression of FASTK during apoptosis signaling pathways has been reported (Brutsche, Brutsche et al. 2001). A close orthologue of this gene is reported in mouse but a low (~27%) identity between these genesit is not sufficient to confidently assign a kinase function to the mouse orthologue.
1.6.8.10 G11 kinase:This family contains a single gene called G11/STK19, which has been shown to bind ATP, and have serine/threonine kinase activity against alpha casein as substrate with a putative catalytic lysine shown as a requirement for enzyme function (Gomez-Escobar, Chou et al. 1998). Homologs of this kinase are found in rat and mouse (~85% identity), and adivergent putative homolog has also been identified in zebrafish (45% identity) with no other homolog seen in any other model organism.
1.6.8.11 Transcriptional Intermediary Factor 1 family:Three Transcriptional Intermediary Factor 1 genes have been grouped in this family, out of which only this kinase has been demonstrated to have kinase activity, in vitro(Fraser at al, 1998). The other two genes are nearly similar and are also likely to be kinases. Drosophila and mosquito genomes also contain a single copy of this gene. Auto-phosphorylation was detected in immune-purified proteins and is believed to be involved in the transcriptional machinery by phosphorylating several other TATA- associated factors. Like TAFs and BRD kinases, the TIFs also contain bromodomains.
1.6.8.12 H11 kinases:The H11 kinase is homologous to the ICP10 gene of Herpes simplex virus, both having distinct kinase activity (Nelson, Zhu et al. 1996, Smith, Yu et al. 2000). Kinase activity of H11was demonstrated in proteins purified from both bacterial and eukaryoticexpression systems. Mutation of a putative catalytic lysine destroyed the kinase activity.
1.6.8.13 RIO family of kinases:The RIO family has 3 distinct subfamilies, with at least a single member of each subfamily in Drosophila, Human and C.elegans. S. cerevisiae has two members, one of each of RIO1 and RIO2 kinases and the fungi Aspergillusnidulanshas a single member among the third subfamily, RIO3. Structural homologs of RIO kinases are also present in many archeal genomes. The first biochemically characterized, Yeast RIO1, was reported to have ser kinase activity (Angermayr, Roidl et al. 2002). Their sequences do not align to a great extent with known eukaryotic protein kinases.Nevertheless,several critical catalytic residues required for kinase activity are conserved in the RIO family too.