Cyclin-dependent kinase homologues of Plasmodium falciparum

International Journal for Parasitology 32 (2002) 1575–1585 www.parasitology-online.com

Invited Review

Cyclin-dependent kinase homologues of Plasmodium falciparum Christian Doerig a, Jane Endicott b, Debopam Chakrabarti c,* a

INSERM U511 team, Wellcome Centre for Molecular Parasitology, The Anderson College, 56 Dumbarton Road, Glasgow G11 6NU, Scotland, UK b Laboratory of Molecular Biophysics and Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, UK c Department of Molecular Biology and Microbiology, University of Central Florida, 12722 Research Parkway, Orlando, FL 32826, USA Received 13 June 2002; received in revised form 5 August 2002; accepted 13 August 2002

Abstract The intraerythrocytic asexual cycle of the malarial parasite is complex and atypical: during schizogony the parasite undergoes multiple rounds of DNA replication and asynchronous nuclear division without cytokinesis. This cell cycle deviates from the classical eukaryotic cell cycle model where, ‘DNA replicates only once per cell cycle’. A clear understanding of the molecular switches that control this unusual developmental cycle would be of great interest, both in terms of fundamental Plasmodium biology and in terms of novel potential drug target identification. In recent years considerable effort has been made to identify the malarial orthologues of the cyclin-dependent kinases, which are key regulators of the orderly progression of the eukaryotic cell cycle. This review focuses on the current state-of-knowledge of Plasmodium falciparum cyclin-dependent kinase-like kinases and their regulators. q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Plasmodium falciparum; Malaria; Cell cycle; Cyclin-dependent kinases

1. Introduction The intraerythrocytic developmental cycle of the malarial parasite diverges from the typical eukaryotic cell cycle where the chromosomes replicate only once. During schizogony, the parasite undergoes multiple rounds of DNA replication to yield polyploidity of chromosomal DNA. This form of nuclear polyploidisation is reminiscent of endoreduplication cycles found in some plant and animal cells (Edgar and Orr-Weaver, 2001). Evidently cells that employ cycles of endoreduplication use a variation of the sequential progression of the classical cell cycle phases of G1, S, G2, and M. Progression of the eukaryotic cell cycle is controlled by a family of protein kinases, the cyclin-dependent kinases (CDKs), whose active forms are composed of a catalytic subunit (CDK) and a regulatory subunit (cyclin) (Morgan, 1997). The temporary association of these subunits, together with the phosphorylation state of the CDK, results in welldefined time windows during which a given CDK is active and phosphorylates its substrates. The CDKs are master regulators that orchestrate the activities of numerous proteins to precisely execute various cell cycle events. Several CDKs and cyclins coexist in eukaryotic cells, with

given combinations being responsible for progression of the cell cycle through particular phases. Transition from one cell cycle stage to the next is linked to the oscillation of CDK activity. A naı¨ve explanation for the endoreduplication cell cycle is that M-CDK activity is inhibited and SCDK activity oscillates to promote multiple rounds of DNA replication. However, since during Plasmodium asexual multiplication asynchronous nuclear divisions appear to intervene between rounds of DNA replication (Read et al., 1993; Arnot and Gull, 1998; Doerig et al., 2000), it is likely that the actual molecular mechanisms regulating erythrocytic schizogony are vastly more complicated. Regardless of the unique characteristics of the Plasmodium cell cycle, it is expected that CDKs will be key molecular switches for cell cycle progression in the parasite. Homologues of several genes, including CDKs and cyclins, that encode critical regulators of DNA replication and cell cycle progression in other model systems, have been identified in Plasmodium falciparum (Table 1). This review focuses on the current state of knowledge of Plasmodium CDK-like kinases and their regulators.

* Corresponding author. Tel.: 11-407-384-2061; fax: 11-407-384-2062. E-mail address: [email protected] (D. Chakrabarti). 0020-7519/02/$20.00 q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(02)00186-8

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Gene

Maximal homology to (% identity over the catalytic domain)

Cyclin-binding motif

PfPK5 PfPK6 Pfmrk

T 14Y 15 T 160 Activity in vitro

CDK1 (cdc2) and CDK5 (60%) PSTTIRE (PSTAIRE in CDK1) CDK1:/MAPKs SKCILRE CDK7 (46%) NFVLLRE (NRTALRE in CDK7) Pfcrk-1 p58-GTA (42%) AMTSLRE (PITSLRE in p58)

TY TY SY

T T T

TY

T

Pfcrk-3 CDK1

AKTYIRE

AY

Pfcrk-4 CDKs MAPK

EEFAVNE

VY

Expression in asexual stages

Expression in gametocytes References

T

Yes; cyclin-dependent Yes (mRNA and protein) Yes; cyclin-independent Yes (mRNA and protein) Yes; cyclin-dependent Yes (mRNA), but mRNA less abundant than in gametocytes ND mRNA not detectable by Northern blot ND Yes (mRNA)

D

ND

Yes (mRNA)

Yes (mRNA)

ND ND Yes (mRNA)

Ross-MacDonald et al., 1994 Bracchi-Ricard et al., 2000 Li et al., 1996

Yes (mRNA)

Doerig et al., 1995

Yes (mRNA)

PlasmoDb and Doerig et al., unpublished PlasmoDb and Doerig et al., unpublished

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Table 1 Plasmodium falciparum cyclin-dependent kinase-like kinases

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2. Structures of CDKs 2.1. CDK activation: cyclin binding and activation loop phosphorylation Structural principles underlying CDK regulation have been elucidated following the determination of a series of CDK and cyclin structures using X-ray crystallography and NMR methods (reviewed in Pavletich, 1999 and Endicott et al., 1999). Though studies on human CDK2 have provided a paradigm for the CDK family, the structures of other CDKs have shown that their differences in activation and regulation are reflected in structural diversity. Plasmodium falciparum CDKs display 40–60% identity with CDKs from other eukaryotes over the kinase domain. Several carry large extensions and even insertions within the catalytic domain, an observation that suggests that the P. falciparum CDK family has unique regulatory features that may be elaborated by their structure determination. Monomeric, unphosphorylated CDK2 (CDK2) adopts the characteristic protein kinase fold (Fig. 1), but is catalytically inactive as a result of the disposition of two key structural elements (De Bondt et al., 1993). Briefly, the C-helix that contains the PSTAIRE motif (single letter amino acid code, residues 45–51), is displaced out from the main body of the protein so that the salt bridge between Glu51 (the E of the PSTAIRE sequence) and Lys33, that is important for correct alignment of the ATP triphosphate moiety for catalysis, is lost (De Bondt et al., 1993). The activation segment (residues 145–172 between and including the conserved DFG and APE motifs) is folded so that the side-chain of Thr160 (the site of activatory phosphorylation) is buried away from solvent and in a conformation that precludes substrate binding.

Fig. 1. Cyclin-dependent kinase (CDK) activation. The structures of monomeric CDK2 (PDB code 1HCK) (a) and T160pCDK2/cyclin A complexed with a peptide substrate (PDB code 1qmz) (b) were superposed using the CDK2 C-terminal lobe. The CDK2 and cyclin folds are drawn in ribbon representation. The CDK2 N- and C-terminal lobes are coloured white and gold, respectively. The cyclin subunit is green. The C-helix (residues 45– 55) is highlighted in magenta and the activation loop (residues 145–172) in cyan. AMP-PNP and the substrate peptide (HHASPRK) are drawn in ball and stick representation with carbon atoms in green, nitrogens in blue and oxygen atoms coloured red. The figure highlights the large structural rearrangements within CDK2, particularly of the C-helix and of the activation loop that accompany cyclin binding and phosphorylation of Thr160.

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CDK2 association with cyclin A, whose interaction with the kinase subunit involves the PSTAIRE helix, induces considerable structural change within the CDK2 active site: catalytically important residues necessary for productive Mg-ATP binding and phospho-transfer are appropriately reorientated and the blockade of the active site by the activation segment is relieved (Jeffrey et al., 1995) (Fig. 1). Thr160, which was previously buried within the CDK2 fold is now exposed to solvent, a change that has been predicted to contribute to its enhanced recognition by CDK7/cyclin H (Kaldis et al., 1998). Phosphorylation of Thr160 (Thr160p) results in a subtle rearrangement of the activation segment that creates the substrate binding site and as a result the activity of the enzyme increases circa 1,000-fold (Hagopian et al., 2001; Russo et al., 1996a,b). CDKs 1, 2, 4 and 6 phosphorylate the consensus sequence S/ T-P-X-K/R, where S/T represents the phosphorylated residue and X represents any amino acid. The structure of a Thr160pCDK2/cyclin A/AMP-PNP/substrate peptide complex has shown that the substrate peptide (HHASPRK) binds in an extended conformation across the kinase surface, primarily contacting the activation segment (Brown et al., 1999). Features of the CDK activation loop conformation dictate CDK specificity for a proline residue at the P 1 1 position and a positively charged residue at P 1 3. The structures of cyclin H (Kim et al., 1996; Andersen et al., 1997), Vcyclin (a cyclin from herpesvirus saimiri) (Schulze-Gahmen et al., 1999), Mcyclin (a g-herpesvirus cyclin), complexed with CDK2 (Card et al., 2000), and Kcyclin bound to CDK6 and p18 ink4c (Jeffrey et al., 2000) have shown that the cyclin box fold is a conserved structural element in the cyclin family despite its lack of sequence conservation. These cyclins, like cyclin A all have additional secondary structural elements outside their cyclin box folds. The structures of CDK2/Mcyclin (Card et al., 2000), CDK5/p25 nck5a (Tarricone et al., 2001) and CDK6/ Vcyclin (Schulze-Gahmen and Kim, 2002) have revealed structural differences between members of the CDK and cyclin families that may reflect differences in their activities and regulation. The structures of these CDK/cyclin complexes suggest that structural changes to both CDKs and cyclins can accompany stable complex formation. Although structures for other monomeric CDKs are not available it is predicted that cyclin association with their cognate CDKs will induce similar changes to the CDK fold as that observed on CDK2 association with either cyclin A or Mcyclin. CDKs and cyclins show distinct preferences for particular partners. The CDK6/Vcyclin interface, though sharing characteristics with that of CDK2/cyclin A also has distinctive features that may explain this cyclin’s observed preference for binding to CDK6 (Schulze-Gahmen and Kim, 2002). Modelling of a CDK2/Vcyclin complex suggested that there would be unfavourably close contacts between the two proteins, an observation that provides at least a partial

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explanation for the inability of this CDK-cyclin pair to associate. Interestingly, CDK2 and cyclin M do not have the same relative disposition as CDK2 and cyclin A and, as a result, structurally equivalent cyclin residues make different interactions with CDK2 (Card et al., 2000). Presumably this altered interface reflects a slightly different optimal packing arrangement as the flexible CDK2 responds to an alternative template. The unique single cyclin box fold of the cyclin p25 nck5a presents a novel cyclin face to CDK5 that can in part explain its ability to bind to a single CDK partner (Tarricone et al., 2001). Such studies, however, are confounded by the plasticity of the CDK fold. Furthermore, within the cell, spatial and temporal factors together with the potential to modulate CDK-cyclin interactions by their association with additional protein partners imply that CDK-cyclin specificity must be the result of multiple contributing factors. As structural studies on the extended CDK family expand it is becoming clear that both cyclin binding and activation loop phosphorylation are not always required for full CDK activation. For example, CDK6 can be fully activated by binding to Vcyclin (Schulze-Gahmen and Kim, 2002) and CDK5 by binding p25 nck5a (Tarricone et al., 2001). The CDK5 activation loop contributes to a substrate binding cleft that is proline-directed and is very similar to that previously observed in a fully activated complex of phosphorylated CDK2/cyclin A bound to a peptide substrate (Brown et al., 1999). CDK1 and CDK2 can be activated in the absence of phosphorylation by binding to members of the RINGO/Speedy family (Karaiskou et al., 2001; Porter et al., 2002). Only the Saccharomyces cerevisiae CDK activating kinase, Civ1, that is a very distantly related CDK family member, has been shown to be active as a monomer (Thuret et al., 1996; Kaldis et al., 1996; Espinoza et al., 1996). The discovery that P. falciparum PfPK6 is also active as a monomer (see subsequently) suggests that more distant relatives within the CDK family may have evolved distinct non-cyclin-dependent mechanisms of activation that could be illuminated by structural analysis. 2.2. CDK association with regulatory proteins Protein association controls CDK activity and structures have been determined to date of CDK2 bound to its regulators p27 Kip1, Cks1 and kinase-associated phosphatase and of CDK6 bound to members of the INK family. The CDK inhibitors fall into two distinct families, the Cip/Kip class and the INKs, that have characteristic preferences for binding to CDKs and are structurally distinct (reviewed in Pavletich, 1999; Endicott et al., 1999). Members of the INK family preferentially bind to monomeric CDK4 and CDK6 whereas Kip/Cip family members bind to both the cyclin and CDK subunits (reviewed in Sherr and Roberts, 1999). INKs contain multiple ankyrin repeats, a motif previously shown to be involved in protein–protein interactions (reviewed in Bork, 1993). The overall fold of CDK6 in

both the CDK6/p16 INK4a (Russo et al., 1998) and CDK6/ p19 INK4d (Brotherton et al., 1998) structures is very similar to that of CDK2 (Fig. 2a) but the result of INK4 association is to distort the ATP binding site so that key residues required for ATP binding and catalysis are misaligned. The CDK6 activation loops also diverge. Members of the Cip/Kip family inhibit CDKs by binding to both CDK and cyclin subunits (Russo et al., 1996a,b). The structure of a ternary p27 Kip1/T160pCDK2/cyclin A complex revealed that p27 Kip1 binds to the cyclin A recruitment site through an N-terminal RXL motif (Adams et al., 1999) and then adopts an a-helical structure as it progresses over the surface of the N-terminal cyclin box fold towards the CDK2 N-terminal lobe (Russo et al., 1998), (Fig. 2b). The inhibitor then forms a b-strand that supplants the edge strand of the CDK2 5-stranded N-terminal b-sheet, and having disrupted the CDK2 fold in this way, finishes off with a 310-helix which binds into the catalytic cleft and blocks ATP binding. A structure for CDK2 complexed with Cks1, a CDK regulatory protein has also been reported (Bourne et al., 1996). Saccharomyces pombe Suc1 was the first member of the Cks family to be identified through its ability when over-expressed to suppress certain CDK1 temperaturesensitive mutations (Hayles et al., 1986). Since then closely related homologues have been identified in a number of species (reviewed in Pines, 1996), but, so far, not in Plasmodium (see subsequently). Following the determination of structures for human Cks2 (Parge et al., 1993) and S. pombe Suc1 (Endicott et al., 1995) two potential sites of Cksprotein interaction were proposed. One of these sites has since been shown to make a substantial contribution to the CDK2/Cks1 interface (Bourne et al., 1996) (Fig. 2c) and the second, ‘charged cluster’ site has been proposed to contribute to a phospho-amino acid binding site (Landrieu et al., 2001). Saccharomyces pombe Suc1 has been shown to stimulate CDK1/cyclin B activity against Cdc25 and Myt1 and Wee1, the phosphatase and kinases, respectively, that are responsible for regulating the extent of CDK1 phosphorylation within the active site (Patra et al., 1999). It may also stimulate CDK1/cyclin B activity towards other substrates later during mitosis (Patra and Dunphy, 1998; Shteinberg and Hershko, 1999). CDKs are tightly regulated by phosphorylation and a structure of CDK2 phosphorylated on Thr160 complexed with CDK-associated phosphatase was the first to reveal the interactions between a phosphatase and its protein substrate (Song et al., 2001), (Fig. 2d). KAP is similar to members of the Cdc25 phosphatase family in that it has very tight substrate selectivity and will only dephosphorylate monomeric CDK2 phosphorylated on Thr160 (Poon and Hunter, 1995). The CDK2/kinase-associated phosphatase complex structure shows that the phosphorylated Thr160 residue is at the tip of an exposed loop and that there are few interactions with kinase-associated phosphatase in this part of the structure. The major protein interface is an exten-

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Fig. 2. Cyclin-dependent kinase (CDK) complexes. A comparison of the structures of selected CDK-containing complexes determined by X-ray crystallography; (a) CDK6/p19 INK4d (PDB code 1BLX), (b) CDK2/cyclin A/p27 Kip1 (PDB code 1JSU), (c) CDK2/Cks1 (PDB code 1BUH), (d) CDK2/KAP (PDB code 1FQ1). The C-terminal domains of CDK2 and CDK6 were superimposed and the four structures are drawn from the same view. To aid comparison, the CDK2 and CDK6 structures are rendered in the same colour scheme as in Fig. 1 (CDK6 activation loop residues 163–189, and C-helix residues 55–65), and the cyclin subunit is coloured mint in panel (b). The p19 INK4d (panel a), p27 Kip1 (panel b), Cks1 (panel c) and KAP (panel d) proteins are rendered in green.

sive one between the kinase-associated phosphatase C-terminal helix and the CDK2 C-terminal lobe and overlaps in part with the CDK2 Cks1 binding site. The determination of additional CDK complex structures will continue to contribute to our understanding of the mechanisms that regulate CDK activity. It is not known how far results from structural studies on higher eukaryotes can be extrapolated to the P. falciparum CDK family. However, they certainly provide a starting point from which to guide structural studies. The unique structural properties of the P. falciparum CDK family will excite much interest in the hope that they will provide a basis for rational drug design.

3. PfPK5 The first CDK-related protein kinase to be reported in Plasmodium was PfPK5. This enzyme was identified following a PCR approach (Ross-Macdonald et al., 1994), and displays similar levels of homology (approximately 60% amino acid identity) to CDK1 and CDK5. It possesses

a fairly conserved PSTAIRE motif (PSTTIRE), and has potential sites of regulatory phosphorylation equivalent to CDK2 Thr14, Tyr15 and Thr160. The 33 kDa protein was found to be expressed throughout erythrocytic schizogony, and immunoprecipitation experiments using extracts from synchronised parasites showed that PfPK5 activity peaks at the schizont stage (36 h post invasion) (Graeser et al., 1996a,b). Interestingly, monomeric recombinant PfPK5 was reported to display low levels of histone H1 kinase activity, a property that had not been previously observed for monomeric CDKs. It was later shown that PfPK5 activity is dramatically increased by addition of cyclins (Le Roch et al., 2000). The first cyclin to be identified as capable of activating PfPK5 in vitro was human cyclin H. Cyclin H had been added to the reaction mixture as a cofactor for Pfmrk (a CDK7 putative homologue, see subsequently) in an attempt to determine whether PfPK5 was a substrate for the Pfmrk-cyclin H complex. Unexpectedly, the control reaction containing only PfPK5 and cyclin H (but no Pfmrk) displayed a level of activity, two to three orders of magnitude higher than that of PfPK5 alone. It was subse-

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quently shown that PfPK5 can be activated in a CAK-independent way by a wide range of cyclins and cyclin-related proteins, including human cyclin H (an otherwise specific CDK7 activator) (Le Roch et al., 2000), p25 (an otherwise specific CDK5 activator) (Le Roch et al., 2000), bovine cyclin A (an otherwise specific CDK1/CDK2 activator) (Holton and Endicott, unpublished), and Pfcyc-1, the first Plasmodium cyclin to be identified (see subsequently) (Le Roch et al., 2000). It is to be noted that PfPK5 is able to autophosphorylate in the presence of a cyclin, another peculiarity that has not been documented for other eukaryotic CDKs. Autophosphorylation does not involve Thr158 (the equivalent of CDK2 Thr160), as a Thr158Ala mutant retains this ability (Holton and Endicott, unpublished). The apparent promiscuity of PfPK5 with regard to multiple cyclins might be explained, at least in part, by the flexibility of the kinase subunit, as has been observed to some degree with CDK2 (see previously). In the case of PfPK5, it can be hypothesised that a more flexible fold might provide an opportunity for subtly different binding modes to be adopted that result in the kinase’s activation.

4. PfPK6 PfPK6 was identified initially by differential display RTPCR of mRNA samples from parasites undergoing transition from ring to schizonts (Bracchi-Ricard et al., 2000). The PfPK6 sequence was also found independently using PCR and primers against conserved sequences within the kinase domain (Doerig, unpublished observation). Despite similar levels of homology to both CDKs and mitogen-associated protein kinases (MAPKs), molecular modelling suggests that PfPK6 might be more closely related structurally to the CDKs. Although the PfPK6 sequence of TVVTLWY in subdomain VIII is similar to that of the Cdc2/CDC28 family EV/IVTLWY consensus sequence, the canonical cyclin binding PSTAIRE motif is replaced by a SKCILRE sequence. The sites of regulatory phosphorylation, found in other CDKs (Thr14, Tyr15 and Thr160 in CDK2) are conserved in PfPK6. A PfPK6 model has been constructed using as the template the structure of active CDK2 extracted from the structure of the phosphorylated CDK2/cyclin A complex (PDB code 1JST) (Russo et al., 1996a,b) (Fig. 3). The PfPK6 sequence is more compatible with the active or partially active structure of CDK2 (PDB code 1FIN, Jeffrey et al., 1995) rather than that of inactive CDK2 (PDB code 1HCL, De Bondt et al., 1993) (data not shown). Several lines of evidence suggests that PfPK6 is a cyclinindependent kinase: (a) the recombinant protein shows significant autophosphorylation and histone phosphorylation activity in the absence of cyclin and (b) unlike PfPK5, the PfPK6 activity is not stimulated by incubation with cyclins. By analogy with other well-studied eukaryotic CDKs it would be predicted that full activation of PfPK6

Fig. 3. Molecular model of PfPK6. The model of PfPK6 (in blue) is generated using an active human cyclin-dependent kinase (CDK2, 1JST) (in black) complexed with cyclin (in grey), phosphorylated as a template using the LOOK/GeneMine program (Lee and Irizarry, 2001).

would require phosphorylation of Thr173, the residue homologous to Thr160 of human CDK2. However, mutation of this threonine had no effect on PfPK6 activity. Although a similar mutation in PfPK5 (Thr158Ala) was reported to abolish activity (Graeser et al., 1996a,b), in another study the mutation had no effect on activity (Holton and Endicott, unpublished). Taken together, these observations suggest that PfPK6 is a unique ‘cyclin-independent’ CDK that may only be regulated through synthesis or degradation. The mechanism of PfPK6 cyclin-independent kinase activity is unclear, but presumably the major changes to vertebrate CDKs that are normally induced by cyclin binding must be constitutively present in PfPK6. Interestingly, PfPK6 contains a relatively large insertion (Asp80-Cys94) in the L6 loop which makes this loop about 10 amino acids longer than that of other CDKs. It is possible that this larger than usual L6 loop may contribute to the constitutive activity of PfPK6, although other protein kinases that are active as monomers (such as cAPK) do not have an equivalent insertion. Mutational analysis of PfPK6 has suggested that autophosphorylation at Thr178 may activate PfPK6 even further through an improvement of substrate binding without affecting ATP binding (Bracchi-Ricard et al., 2000), a role analogous to that of CDK2 Thr160 (Hagopian et al., 2001). The simultaneous loss of autophosphorylation activity and substrate phosphorylation (ribonucleotide reductase small subunit R2 kinase activity) in the PfPK6 Thr178Ala mutant suggests, albeit does not establish, that this threonine

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Fig. 4. Magnification of the catalytic region of interest in PfPK6 and its mutants. The CARA module of the LOOK program was used to model the wild-type (a) and each of the three mutant versions of PfPK6: Thr178Ala, Thr178Asp and Thr178Glu (b–d). The side chain of Thr178 (or the corresponding mutation) in the variable ‘T’ loop (in green) is shown as well as the side chains of the two highly conserved residues Lys145 and Asp143 in the catalytic loop (in red). The possible hydrogen bonding between Thr178 (or Ala178, Asp178, Glu178) and Lys145 and/or Asp143 are indicated in blue.

may be a major site of autophosphorylation and that autophosphorylation is important for full activation of PfPK6. If this is true, PfPK6 could potentially be regulated by dephosphorylation by a P. falciparum phosphatase that remains to be characterised. It is of interest to note that mutations of Thr178 to a glutamate or an aspartate (which can in some instances mimic phosphorylated threonine or serine) did not restore PfPK6 activity. As illustrated in Fig. 4a, the molecular model of the PfPK6 catalytic domain predicts that Thr178 could hydrogen bond to Asp143 in the catalytic loop but that the mutants Thr178Ala, Thr178Glu or Thr178Asp could not (Fig. 4b–d). A similar interaction between threonine and aspartate residues in the catalytic loop has recently been demonstrated for Acanthamoeba myosin I heavy chain kinase (Szczepanowska et al., 1998). PfPK6 exhibits other unusual properties such as its preference for Mn 21 as the divalent cation, a property usually associated with tyrosine kinases. PfPK6 also resembles the MAPKs in that its sensitivity to roscovitine, is in the micromolar rather than the nanomolar range as reported for CDK1, CDK2 or CDK5 (Bracchi-Ricard et al., 2000). It can also efficiently use myelin basic protein as a substrate (Equinet and Doerig, unpublished). Interestingly, the TXY activation site that is conserved in MAPKs, and must be phosphorylated on both the T and the Y residues for the MAPK to become fully active (Garrington and Johnson, 1999), is substituted by TPT in PfPK6. The CDKs possess only one residue that can be phosphorylated in the region, corresponding to Thr160 in CDK2. It would be informative to determine whether both residues must be phosphorylated for full activation of PfPK6. Clearly, the determination of the structure of PfPK6 will be important to elucidate the mechanism of its cyclin-independent activity and its relationship to MAPKs. The expression of PfPK6 transcript and protein peaks in trophozoites and early schizonts, and several alternately spliced PfPK6 transcripts have been identified. The physio-

logical significance of these variant transcripts is not clear. The major protein in Plasmodium cell extracts that crossreacts with a PfPK6 antibody has a Mr of 35 kDa, a size compatible with it being translated from the properly processed message. Immunofluorescence microscopy has shown that PfPK6 localises both to the nucleus and to the cytoplasm. A search for putative substrate motifs using a combinatorial random dodecapeptide library prepared by fusing peptide 12-mers to pIII coat protein of filamentous coliphage M13 (Ph.D-12e library, New England Biolab) yielded predominantly S/T-P-X-K/R containing peptides. This motif is the consensus CDK phosphorylation site. When a phage display cDNA library constructed in the T7Select vector (Novagen) was used to identify putative PfPK6-interacting proteins, a large number of cDNA fragments were found to derive from elongation factor-1a (EF1a). Interaction of PfPK6 with EF1a was subsequently confirmed by pull-down assays (Bracchi-Ricard and Chakrabarti, unpublished). It is possible that PfPK6 may regulate the cell cycle indirectly through its ability to affect various cellular activities.

5. Pfmrk In higher eukaryotes, CDK7, in conjunction with cyclin H and the Mat1 protein, is the CDK-activating kinase responsible for activating other CDK/cyclin complexes by phosphorylating CDK catalytic subunits on the threonine residue equivalent to CDK2 Thr160. A gene encoding a kinase with 46% identity to human CDK7 and 36–43% identity to other CDKs, was identified by a PCR approach and was called Pfmrk (MO15-related kinase, MO15 being another denomination for CDK7) (Li et al., 1996). Pfmrk possesses a fiveresidue insertion just before the C-helix that is important for cyclin binding (other CDK-activating kinases also display short insertions at this location), and a 13-residue insertion

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within the activation loop. Pfmrk expressed in Escherichia coli displays very little or no HI kinase activity in vitro as a monomer, but can be moderately activated by the presence of human cyclin H (Waters et al., 2000) or Pfcyc-1 (a P. falciparum protein with maximal homology to cyclin H, see subsequently) (Le Roch et al., 2000). It has been subsequently shown that the carboxy-terminal domain of RNAP polymerase II (CTD) is a better substrate than histone H1 for Pfmrk (Li et al., 2001). However, Pfmrk-cyclin complexes have no detectable activity in vitro towards PfPK5 or other plasmodial CDK-related kinases. Whether this result reflects the fact that Pfmrk is not a CDK-activating kinase but has entirely different functions in the parasite (in addition to its CDK activating kinase activity, mammalian CDK7 is implicated in other functions such as transcriptional regulation), or whether no CDK-activating kinase activity is detectable because one or more accessory elements (such as a possible Mat1 functional homologue) are lacking in the assays, is unclear at present. Pfmrk/Pfcyc1 is similar to PfPK5 in that it is inhibited by p21 Cip1 and not by p16 INK4 (Li et al., 2001). Pfmrk mRNA was detected in both asexual parasites and gametocytes, with apparently higher levels in the latter.

6. Other CDK-related kinases in the P. falciparum genome: Pfcrk-1, Pfcrk-3 and Pfcrk-4 Pfcrk-1 was identified by a PCR approach that aimed to identify a CDK1 homologue using degenerate primers (Doerig et al., 1995). The Pfcrk-1 ORF was found to display maximal homology to the p58-GTA ‘PISTLRE’ family. This group of kinases with high homology to CDKs appear to be implicated, in higher eukaryotes, in a variety of functions including apoptosis and cell differentiation. For example, during early mouse embryogenesis, isoforms of PISTLRE kinases are expressed in populations which withdraw from proliferation and undergo differentiation (Lahti et al., 1995). To our knowledge, no cyclin partner has yet been identified for these enzymes. Pfcrk-1 mRNA is detectable in gametocytes but not in asexual blood stages, outlining an expression pattern broadly similar, in terms of cell division, to that of PISTLRE isoforms. The availability of genomic databases has allowed the identification of Pfcrk-3, a gene encoding a putative kinase with maximal homology to CDK1, and of Pfcrk-4, which, like PfPK6, displays similar levels of homology to both CDKs and MAPKs. Both these ORFs are expressed in asexual and sexual blood stages (Le Roch, Merckx, Equinet, Doerig, unpublished). A threonine equivalent to CDK2 Thr160 appears to be present in all three ORFs, although in the case of Pfcrk-4 insertions within the activation loop make the alignment difficult. The residues corresponding to Thr14 and Tyr15 of CDK2 are conserved in Pfcrk-1, but in Pfcrk-3 and Pfcrk-4 Thr14 is substituted by an alanine and a valine, respectively. This finding may indicate that these

enzymes have diverged in their ability to be regulated by inhibitory phosphorylation. Pfcrk-1, Pfcrk-3 and Pfcrk-4 all share the peculiarity of possessing a very large (several hundred amino-acids) extension at their N-terminus, which contains repeated motifs as found in many other types of Plasmodium proteins. It is unclear at this stage whether these extensions are involved in enzyme function, or whether they are processed during the infection. In addition to this extension, Pfcrk-3 and Pfcrk-4 also carry large insertions within their catalytic domains. No function has yet been demonstrated for Pfcrk-1, -3 and -4.

7. Cyclins and other CDK regulators in Plasmodium Despite attempts by several groups to identify cyclins by PCR-based approaches using conserved sequences within the cyclin box, a malarial cyclin gene was not reported until the genomic databases became available. Database mining using cyclin sequences as queries led to the identification of an ORF called Pfcyc-1, which displayed maximal homology to cyclin H from a variety of organisms (Le Roch et al., 2000). The low level of homology (approximately 17%) of Pfcyc-1 with mammalian cyclin H probably explains the failure of cloning attempts based on PCR with degenerate primers. It came as a surprise that bacterially expressed Pfcyc-1 was in vitro able to efficiently activate PfPK5 (see previously). This result suggested that PfPK5 may have a relaxed specificity in its cyclin requirements, a hypothesis that was confirmed by the demonstration that PfPK5 can be activated by other cyclins and cyclinlike proteins such as p25, a CDK5 activator protein structurally related to cyclins despite a lack of homology at the sequence level. Pfcyc-1 also activates Pfmrk, which was somewhat less surprising since Pfcyc-1 displays maximal homology to cyclin H and Pfmrk to CDK7. Nevertheless, this result illustrates the difficulty in predicting the function of regulatory elements from their sequence. At least three additional ORFs encoding cyclin-related proteins have been located in PlasmoDB and are under investigation. As was the case for cyclins, PCR-based searches for CDK inhibitors have not lead to the identification of genes encoding such elements in Plasmodium. Furthermore, sequencebased searches of the P. falciparum genome have failed so far to identify any putative P. falciparum proteins with significant sequence homology to any CDK inhibitor homologues. It has been reported that PfPK5/Pfcyc-1 and Pfmrk/ Pfcyc-1, but not PfPK6 can be inhibited by p21 Cip1 (a member of the Cip/Kip class of CDK inhibitors) and not by p16 INK4a (a member of the INK family) in in vitro assays (Li et al., 2001). This result suggests that P. falciparum CDK complexes may be subject to regulation by CDK inhibitors and by analogy with the roles of CDK inhibitors in higher eukaryotic cells the identification of bonafide CDK inhibitors in P. falciparum would suggest the existence of checkpoint pathways to regulate CDK activity. Hence, it

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cannot be excluded that the Plasmodium CDKs are regulated in vivo by CKI functional homologues which are too divergent at the sequence level to allow their identification by in vitro (PCR with degenerate primers) or in silico approaches. This conundrum appears to apply as well to other CDK regulators such as the Wee1 and Myt1 kinases, and the Cdc25 phosphatase. Genes obviously encoding homologues of these elements cannot be found in PlasmoDB among the numerous kinase- and phosphataseencoding genes in the P. falciparum genome. Clearly, alternative experimental approaches (see subsequently) are needed to identify CDK regulators.

8. Future prospects Understanding the role of CDKs in the regulation of the Plasmodium cell cycle requires a two-pronged approach: first, we need to identify the entire complement of CDKs and their positive or negative regulators; and second, the precise function of these elements in cell cycle progression must be determined. Database mining has been already proven as immensely useful in this context. This approach has to be pursued and refined, and will undoubtedly contribute significantly to the identification of additional relevant genes. Nevertheless, we have alluded previously to the limitations of in silico and in vitro approaches to gene identification based solely on sequence homology. The genomic database can be used in another way to identify CDK regulators: proteins from parasite extracts can be purified by affinity chromatography on immobilised tagged kinases or cyclin subunits, and bound proteins can then be analysed by mass spectrometry. The screening of peptide mass spectra against the database should then lead to the identification of these proteins. Furthermore, protein chip technology could also be used to identify biochemical properties of Plasmodium protein kinases similar to that reported for yeast protein kinases (Zhu et al., 2000). Functional studies of CDKs are hampered by the fact that these enzymes are very likely essential for the completion of the asexual erythrocytic cycle. This means that it is not feasible to adopt a classical knock-out approach to investigate null phenotypes, since a selection step during erythrocytic schizogony is required to obtain mutant parasites. A possible solution to this problem may lie in a ‘chemical genetics’ approach, where a mutation is introduced into the kinase that does not result in a loss-of-function. This technique makes the preparation of mutant parasite lines feasible even if the targeted gene is essential by generating a kinase that is hypersensitive to inhibitors that do not affect the wild-type enzyme (Bishop et al., 2001). If the wild-type allele is replaced in the parasite by such a mutant allele, a null phenotype can in principle be obtained by the treatment of the parasite with the inhibitor. These new approaches should shed some light on the

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molecular mechanisms regulating the exquisitely atypical cell cycle of malarial parasites. Characterisation of the critical factors required for progression of the malarial parasite cell cycle will not only be valuable in understanding the evolutionary conservation of their unique functions but will also lead to the definition and/or validation of potential novel drug targets among regulators of malarial cell proliferation. Acknowledgements Work in C.D.’s laboratory is supported by INSERM, The French Ministries of Research (PAL 1 and PRFMMIP programmes), Defence (De´ le´ gation Ge´ ne´ rale pour l’Armement), Education, and Foreign Affairs (French-South African Programme for Cooperation in Science and Technology), and by the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR). The work in J.A.E.’s laboratory is supported by the MRC, BBSRC, Oxford University, The Royal Society and The Wellcome Trust. J.A.E. would like to thank Martin Noble and Simon Holton for their many contributions and Martin Noble for his assistance in the preparation of the figures. The research in D.C.’s laboratory is supported by a grant from the National Institutes of Health (AI48036). References Adams, P.D., Li, X., Sellers, W.R., Baker, K.B., Leng, X., Harper, J.W., Taya, Y., Kaelin Jr, W.G., 1999. Retinoblastoma protein contains a Cterminal motif that targets it for phosphorylation by cyclin-cdk complexes. Mol. Cell. Biol. 19, 1068–80. Andersen, G., Busso, D., Poterszman, A., Hwang, J.R., Wurtz, J.-M., Ripp, R., Thierry, J.-C., Egly, J.-M., Moras, D., 1997. The structure of cyclin H: common mode of kinase activation and specific features. EMBO J. 16, 958–67. Arnot, D.E., Gull, K., 1998. The Plasmodium cell-cycle: facts and questions. Ann. Trop. Med. Parasitol. 92 (4), 361–5. Bishop, A.C., Buzko, O., Shokat, K.M., 2001. Magic bullets for protein kinases. Trends Cell Biol. 11 (4), 167–72. Bork, P., 1993. Hundreds of ankyrin-like repeats in functionally diverse proteins: mobile modules that cross phyla horizontally? Proteins 17, 363–74. Bourne, Y., Watson, M.H., Hickey, M.J., Holmes, W., Rocque, W., Reed, S.I., Tainer, J.A., 1996. Crystal structure and mutational analysis of the human CDK2 kinase complex with cell cycle-regulatory protein CksHs1. Cell 84, 863–74. Bracchi-Ricard, V., Barik, S., Delvecchio, C., Doerig, C., Chakrabarti, R., Chakrabarti, D., 2000. PfPK6, a novel cyclin-dependent kinase/mitogen-activated protein kinase-related protein kinase from Plasmodium falciparum. Biochem. J. 347 (Pt 1), 255–63. Brotherton, D.H., Dhanaraj, V., Wick, S., Brizuela, L., Domaille, P.J., Volyanik, E., Xu, X., Parisini, E., Smith, B.O., Archer, S.J., Serrano, M., Brenner, S.L., Blundell, T.L., Laue, E.D., 1998. Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell cycle inhibitor p19 INK4d. Nature 395, 244–50. Brown, N.R., Noble, M.E.M., Endicott, J.A., Johnson, L.N., 1999. The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases. Nat. Struct. Biol. 1, 438–43. Card, G.L., Knowles, P., Laman, H., Jones, N., McDonald, N.Q., 2000.

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