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Isolation and characterization of a cdc2-related protein kinase 3 (CRK3) from a

Sudanese strain of Leishmania donovani

Article · September 2012

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International Research Journal of Pharmacy and Pharmacology (ISSN 2251-0176) Vol. 2(9) pp. 215-224, September 2012
Available online http://www.interesjournals.org/IRJPP
Copyright © 2012 International Research Journals

Full Length Research Paper

Isolation and characterization of a cdc2-related protein

kinase 3 (CRK3) from a Sudanese strain of Leishmania
donovani
Nahla O. M. Ali 1, 2*, Muntaser E. Ibrahim3, Hamid S. Abdalla1, Moawia M. Mukhtar3,
Ahmed M. El-Hassan4, Jeremy C. Mottram2
1
Department of Parasitology, Faculty of Veterinary Medicine, University of Khartoum, SUDAN.
2
Wellcome Trust Centre for Molecular Parasitology, Glasgow Biomedical Research Centre, University of Glasgow,
Scotland, UK.
3
Department of Molecular Biology, Institute of Endemic Diseases, University of Khartoum, SUDAN.
4
Department of Epidemiology and Clinical Science, Institute of Endemic Diseases, University of Khartoum, SUDAN.
Accepted 17 July, 2012

Cyclin-dependent kinases; (cdc2) are key regulators of the eukaryotic cell cycle. A number of cdc2-
related kinase (CRK) genes have been isolated from trypanosomatids. The present study was designed
to identify and analyze (CRK3) from a Sudanese strain of Leishmania donovani, the causative agent for
Kala-azar. CRK3. Sequence analysis showed that L. donovani CRK3 encodes a protein of 311 amino
acids with 99.7%; 99.4% and 49.4% identity with L. mexicana CRK3; L. major CRK3 and human HsCDC2,
respectively. Genetic analysis by southern blot hybridisation of the genomic DNA has demonstrated
that LdCRK3 gene is not tandemly repeated and is present as a single copy in the L. donovani genome.
Phylogenetic analysis showed that all of the three leishmanial CRK3s proteins exist with the
trypanosome CRK3s in the same clade. CRK3 has potential as drug target, so the scientific data
presented in this paper could be useful to explore the possibility of designing a drug that can be used
in a low-cost manner to treat all forms of leishmaniasis based on the fact that CRK3 is highly conserved
in Leishmania spp.

Keywords: Leishmania donovani, CRK3, protein kinase, molecular cloning, Sudan.

INTRODUCTION

Leishmania are parasitic protozoa that have a complex sequence variations in the cytochrome oxidase II gene in
life cycle, during which the parasite shuttles between an assortment of Leishmania isolates has shown that the
rapidly dividing stages (promastigotes and amastigotes) Sudanese isolates of L. donovani possessed the most
and the cell cycle arrested metacyclic stage. Leishmania ancestral sequences and were of a single haplotype that
donovani is the prototype microorganism responsible for significantly resembled the sequence of L. major (Ibrahim
visceral leishmaniasis (VL) in tropical and sub-tropical and Barker, 2001).
regions of the world including the Sudan (Zijlstra and el- There is an inherent link between the control of the
Hassan, 2001). Genetic studies have shown the parasite’s life cycle and cell cycle regulation. Cyclin-
relationship between various genes of both L. donovani dependent kinases (CDKs), exemplified by cdc2 regulate
(causative agent of VL) and L. major (causative agent of progression through the eukaryotic cell cycle (Lee and
cutaneous leishmaniasis [CL]). For instance, analysis of Nurse, 1987). They are also involved in the regulation of
other processes such as gene expression and phosphate
metabolism. Two control points of the cell cycle are
thought to be regulated by the CDKs, the START and the
G2/M transition (Pines, 1995). The latter is highly
* Corresponding Author E-mail: [email protected]; regulated by the activity of the cdc2 gene. A large
Tel: (+) 249 922 579271; Fax: (+) 249 185 312638 number of homologues have been identified from differ-
216 Int. Res. J. Pharm. Pharmacol.

Table 1. Oligonucleotides primers used in this study.

Primer Length Sequence 5′′-3′′ Comments

OL322 (Forward) 25 GGCCATGGCTTCGTTTGGCCGTGTG Pc PCR Cloning of Lmmcrk3
OL323 (Reverse) 27 GCGGATCCCTACCAACGAAGGTCGCTG , ,, ,, ,, ,, ,,

ent eukaryotes, including the unicellular trypanosomatids, oligonucleotides mainly based on the sequence that flank
which were designated cdc2-related kinases rather than the START and the STOP codons of the L. mexicana
CDKs at that time because none of them had been CRK3 gene (Grant., 1998). The PCR-amplified cloned
shown to bind cyclins (Mottram, 1994). The first fragment of L. donovani CRK3 was labelled by random
CRK/cyclin complex identified in T. brucei was the priming to an approximate specific activity of 50 µCi, and
CRK3/CYC2 complex (Van Hellemond et al., 2000). Most was used as a probe to screen a λ Zap Cdna library (9 x
recent study has shown that a recombinant L. mexicana 107 pfu / µl) synthesized from the promastigote stage of
CRK3 and cyclin CYCA were combined in vitro to the L. donovani (Christensen et al., 2000). Two positives
produce an active histone H1 kinase that was inhibited by λ clones were isolated after two rounds of screening,
the CDK inhibitors, flavopiridol and indirubin-3’-monoxime were confirmed by PCR analysis and found to contain the
(Gomes et al., 2010). L. donovani CRK3 gene (LdC1 & LdC2) (Ali et al., 2003).
CRK3 homologues have been described in the
trypanosomatids Leishmania mexicana, LmCRK3 (Grant
et al., 1998), Leishmania major, LmajCRK3 (Wang et al., Excision of the LdC1 and LdC2 phagemids:
1998), Trypanosoma brucei, TbCRK3 (Mottram and
Smith, 1995) and Trypanosoma cruzi, TzCRK3 (Hassan The positive clones (LdC1 & LdC2) were excised with
et al., 2001). CRK3 proteins have been tested for their helper phage to generate subclones in pBlue Script SK (-
ability to complement different Schizosaccharomyces ) phagemid vector. The Rapid Excision kit (Stratagene)
pombe cdc2 and / or Saccharomyces cerevisiae CDC28 was used for this purpose. Briefly, a volume of 200 µl of
temperature sensitive mutants, while, L. mexicana CRK3 XL-1 Blue was co-infected with 250 µl of the cored plaque
failed to complement cdc28ts mutants (Grant et al., 1998).
and 1 µl (1010 pfu/ml) of the ExAssist helper phage and
Only L. major CRK3 was able to restore Cdc2p / CDC28 0
were incubated as usual at 37 C. The excised phagemid
activity (Wang et al., 1998). Taken together, these
was ittered by adding (1-3) µl to 200 µl XLOLR strain of
results suggest that, at least for trypanosomatids and
E. coli and were then plated onto LB-tetracycline (25
yeast, cross-species complementation may not be a
powerful method to establish functional homologies µg/ml) agar plates and incubated overnight at 370C.
between proteins. Gene disruption experiments in L. Purification of λ DNA:
mexicana indicated that LmmCRK3 was essential to the The bacteriophage DNA of the positive clones was
promastigote form, and the histidine tagged CRK3 has purified using Qiagene Miniprep kit (Qiagen). Briefly, a
shown high activity towards the substrate histone H1 that volume of 3 ml LB broth containing the appropriate
was mainly observed at the G2/M, suggesting a possible antibiotics, were inoculated with a single colony and then
role in controlling the cell cycle (Hassan et al., 2001). incubated overnight at 370C in a 200 rpm shaker. The
The aims of this study: firstly, to isolate and suspension, lysis, neutralization, washing, and elution
characterize the full-length CRK3 gene from L. donovani, were all performed as described by the manufacturer.
using PCR cloning, library screening, automated- The DNA was precipitated and quantified as previously
sequencing techniques. Secondly, characterization of described (Ali et al., 2003).
the encoded CRK3 protein, analysis of the structure and Sequencing of the isolated LdC2 clone:
comparison with cdc2 homologs from other organisms, Sequencing internal primers (Table 1) were designed
mainly, Leishmania spp. And the human by using based on sequences obtained by using T3 (forward) and
computer-based sequence analysis programs. T7 (reverse) primers. The purified LdC2 clone was used
as a template in those sequencing reactions. The gene
was identified as a cdc2-related kinase by computer
analysis using the AlignX software, part of Vector NTI
MATERIALS AND METHODS Suite V 6.0.

Isolation of the positive λ phage clones LdC1 & LdC2

Southern Blot Analysis
The probe that was used in this screen was derived from
a PCR product that was produced from L. donovani Six micrograms of L. donovani strain (1s)
(MHOM/SD/63/ ittered) genomic DNA using (MHOM/SD/63/Khartoum) genomic DNA was digested
 Ali et al. 217

with restriction endonucleases EcoRI, HindIII, BglII, serine/threonine protein kinase family, mainly those
BamHI, SalI and NotI., electrophoresed through a which are important for the regulation of cdc2 activity.
0.8% agarose gel, and blotted onto Hybond-N membrane This comprised equivalent residues to human CDC2 at
(Amersham Pharmacia, UK). The blot was hybridized Thr-14 (T14) and Tyr-15 (Y15) in the ATP-binding
32
with a 2 Kb P random-primed HindIII fragment of L. domain, and T161 (Figure 1). The most highly conserved
mexicana crk3 (Containing Lmmcrk3 ORF, Accession regions between HsCDC2 and L. donovani CRK3 include
0
number AJ001275) at 65 C overnight. Washes were for a 10-amino acid block GEGTYGVVYK, in the ATP-
0
20 min at 65 C with 2x SSC / 0.1% SDS and then twice binding region (Figure 1), that is thought to be identical in
for 15 min with 0.1 x SSC / 0.1% SDS. Finally, the all functional CDC2/CDK kinases. However, the L.
membrane was autoradiographed. donovani CRK3 has a single substitution, where the
lysine residue (K) is replaced by arginine residue (R),
interestingly, all the three leishmanial CRK3s have the
RESULTS same substitution. As both amino acids are basic, this
may suggest that no significant differences exist between
Isolation of L. donovani CRK3 these kinases based on the ATP-binding domain. The
16-aa sequences EGVPSTAIREISLLKE known as the
A 900 bp DNA fragment was produced from the genomic ‘PSTAIRE’ box is a highly conserved domain thought to
DNA of the L. donovani, using a set of oligonucleotide be important for the binding of cyclins. In this ‘PSTAIR’
primers (OL322 and OL323) that have been used box six substitutions occurred; in the first one isoluecine
successfully to isolate the L. mexicana CRK3 gene. (I) for valine (V), glutamine (Q) for serine (S) in the
Sequencing of the LdC2 λ clone has shown a nearly full- second substitution, leucine (L) for the isoleucine (I) in
length L. donovani CRK3, as it contains a complete 3′ the third substitution, valine (V) for isoleucine (I) in the
UTR end (data not shown). However, the 5′ UTR end is fourth substitution, isoleucine (I) for leucine (L) in the fifth
incomplete as it lacks the spliced leader (SL). In addition, substitution, and glutamine (Q) for (K) in the sixth
the sequence was found to contain a single ORF, substitution. Two of these substitutions give ‘PQTALR’
translation of which, revealed the presence of an motif instead of ‘PSTAIR’. However, this feature is
encoded sequence of 311 amino acids. Further analysis common in all trypanosomatid CRK3. LdCRK3 also
of the ORF revealed that, as with other leishmanial contains sequence features that distinguish it from the
genes, the L. donovani CRK3-encoding sequence functional CDC2s. The region near the COOH terminus
preferentially preserved a fairly high G or C codon at the is of low homology (subdomain X). L. donovani CRK3
third base of the codon (wobble position). Approximately has a GDSEIGQ motif in place of the highly conserved
51% of the codon used contained either a G or a C GDSEIDQ box (subdomain IX) that plays major role in
residue at the third position. The full sequence of the the control of the phosphorylation at the conserved 161-
LdC2 λ clone, which contains LdCRK3 (L. donovani Thr residue.
cdc2-related kinase 3), has been deposited in the EMBL Sequence identity (%) between the L. donovani CRK3,
(Accession number AJ426472). cdc2-related kinases (CRKs) from other trypanosomatids,
budding yeast CDC28, and human CDC2 has been
investigated (Table 2). L. donovani CRK3 shares over
Characterization of L. donovani CRK3 99% identity with both L. mexicana CRK3 and L. major
CRK3, over 78% identity with T. brucei CRK3 and T.
The predicted protein encoded by the L. donovani CRK3 cruzi CRK3, and over 49% with both human CDC2 and
gene shows the greatest degree of sequence identity to Saccharomyces cerevisiae CDC28. In contrast to L.
the cdc2 family of serine/threonine protein kinases when mexicana CRK3, L. mexicana CRK1 has only 42%
compared with protein sequence databases. The identity with the L. donovani CRK3, which indicates the
alignment of L. donovani CRK3 with L. mexicana CRK3 degree of divergence between the two CRKs, and may
(LmCRK3), L. major CRK3 (LmajCRK3) and human cdc2 also suggest that they play two different roles in the
(HsCDC2) are shown in Figure 1. The alanine residue at parasite Leishmania. The lowest identity is shown by the
position 7 (Ala-7) was replaced by a valine residue (Val- L. mexicana CRK4, to which L. donovani CRK3 has less
7) in both of the L. mexicana CRK3 and L. major CRK3, than 23% identity.
while the conserved alanine residue at position 217 (Ala-
217) in both of the L. donovani CRK3 and L. mexicana
CRK3 was replaced by threonine residue (Thr-217) in the Phylogenetic analysis
L. major CRK3. The three leishmanial CRK3s each have
an unusual 19-amino acid N-terminal extension, when As illustrated in Figure 2, phylogenetic analysis using
compared with human cdc2, which is highly conserved in AlignX program of the L. donovani CRK3 with other cdc2-
sequence. However, all the three leishmanial CRK3s related kinases show that all three leishmanial CRK3s
have the domains and residues characteristic of the proteins are grouped with the trypanosome CRK3s in one
218 Int. Res. J. Pharm. Pharmacol.

ATP-binding
1 ** 50
LdCRK3 (1) MSSFGRATARSGDAGTRDSLDRYNRLDVLGEGTYGVVYRAVDKITGQYVA
LmCRK3 (1) MSSFGRVTARSGDAGTRDSLDRYNRLDVLGEGTYGVVYRAVDKITGQYVA
LmajCRK3 (1) MSSFGRVTARSGDAGTRDSLDRYNRLDVLGEGTYGVVYRAVDKITGQYVA
HsCDC2 (1) -------------------MEDYTKIEKIGEGTYGVVYKGRHKTTGQVVA
PSTAIRE-Box
51 100
LdCRK3 (51) LKKVRLDRTEEGIPQTALREVSILQEFDHPNIVNLLDVICSDGKLYLVFE
LmCRK3 (51) LKKVRLDRTEEGIPQTALREVSILQEFDHPNIVNLLDVICSDGKLYLVFE
LmajCRK3 (51) LKKVRLDRTEEGIPQTALREVSILQEFDHPNIVNLLDVICSDGKLYLVFE
HsCDC2 (32) MKKIRLESEEEGVPSTAIREISLLKELRHPNIVSLQDVLMQDSRLYLIFE

101 150
LdCRK3 (101) YVEADLKKAIEKQEGG--YSGMDLKRLIYQLLDGLYFCHRHRIIHRDLKP
LmCRK3 (101) YVEADLKKAIEKQEGG--YSGMDLKRLIYQLLDGLYFCHRHRIIHRDLKP
LmajCRK3(101) YVEADLKKAIEKQEGG--YSGMDLKRLIYQLLDGLYFCHRHRIIHRDLKP
HsCDC2 (82) FLSMDLKKYLDSIPPGQYMDSSLVKSYLYQILQGIVFCHSRRVLHRDLKP
T161-P
151 * 200
LdCRK3 (149) ANILLTSGNVLKLADFGLARAFQVPMHTYTHEVVTLWYRAPEILLGEKHY
LmCRK3 (149) ANILLTSGNVLKLADFGLARAFQVPMHTYTHEVVTLWYRAPEILLGEKHY
LmajCRK3(149) ANILLTSGNVLKLADFGLARAFQVPMHTYTHEVVTLWYRAPEILLGEKHY
HsCDC2 (132) QNLLIDDKGTIKLADFGLARAFGIPIRVYTHEVVTLWYRSPEVLLGSARY

201 250
LdCRK3 (199) TPAVDMWSVGCIFAELARRKVLFRGDSEIGQLFEIFQVLGTPTDTEGSWP
LmCRK3 (199) TPAVDMWSVGCIFAELARRKVLFRGDSEIGQLFEIFQVLGTPTDTEGSWP
LmajCRK3(199) TPAVDMWSVGCIFAELTRRKVLFRGDSEIGQLFEIFQVLGTPTDTEGSWP
HsCDC2 (182) STPVDIWSIGTIFAELATKKPLFHGDSEIDQLFRIFRALGTPNNE--VWP

251 300
LdCRK3 (249) GVSRLPDYRDVFPKWTAKRLGQVLPELHPDAIDLLSKMLKYDPRERISAK
LmCRK3 (249) GVSRLPDYRDVFPKWTAKRLGQVLPELHPDAIDLLSKMLKYDPRERISAK
LmajCRK3(249) GVSRLPDYRDVFPKWTAKRLGQVLPELHPDAIDLLSKMLKYDPRERISAK
HsCDC2 (230) EVESLQDYKNTFPKWKPGSLASHVKNLDENGLDLLSKMLIYDPAKRISGK

301 318
LdCRK3 (299) EALQHPWFSDLRW-----
LmCRK3 (299) EALQHPWFSDLRW-----
LmajCRK3(299) EALQHPWFSDLRW-----
HsCDC2 (280) MALNHPYFNDLDNQIKKM

Figure 1. Comparison of the Amino Acid Sequence of L. donovani CRK3 (LdCRK3) with L. mexicana CRK3 (LmCRK3), L.
major CRK3 (LmajCRK3) and Human CDC2 (HsCDC2).
Sequences were aligned using AlignXTM software part of the VectorNTI package. Dashes indicate gaps made to maximize
alignments. Tyrosine and Threonine residues (*) shown to be phosphorylated in vivo in vertebrate CDC2s are indicated. The
black shading represents sequences those are identical in human and the parasite Leishmania, grey shading represents
sequences those are identical in all Leishmanial cdc2-related kinase 3 (CRK3). The ATP-binding domain and the PSTAIRE-
Box are illustrated. The numbers between brackets show the start of the amino acid sequence of every protein in each line,
while the other numbers show the start and the end of the amino acid sequence in each line based on the Leishmanial CRK3s
proteins.

Table 2. Sequence identity (%) between L. donovani

CRK3, Trypanosomatid cdc2-related kinases (CRKs),
Budding yeast CDC28, and Human CDC2.

% Identity LdCRK3 HsCDC2 ScCDC28

LdCRK3 - 49.4 50.2
LmajCRK3 99.4 49.1 50.3
LmmCRK3 99.7 49.4 50.2
LmmCRK1 42.5 51.5 50.3
LmmCRK4 22.7 28.8 26.8
TbCRK3 78.1 49.4 47.9
TzCRK3 77.8 48.7 47.9
Reference: HsCDC2 (Accession # X05360), L. mexicana
CRK3 (Accession # AJ001275), L. mexicana CRK1
(Accession # X60385), L. mexicana CRK4 (Accession #
AJ293288), L. major CRK3 (Accession # AF073381), L.
donovani CRK3 (Accession #AJ426472), ScCDC28
(Accession # X00257), T. brucei CRK3 (Accession #
X74617), T. cruzi CRK3 (Accession # U69958).
 Ali et al. 219

Figure 2. Phylogenetic analysis of L. donovani CRK3 with other cdc2-related kinases

using AlignXTM program of the VectorNTI suite.
Catalytic domains were used in this analysis. Species abbreviations are as follows: Hs,
human; Sc, Saccharomyces cerevisiae; Tb, T. brucei; Tz, T. cruzi, Lmm, Leishmania
mexicana; Lmaj, Leishmania major; Ld, Leishmania donovani. Reference: LmmCRK1
(Accession # X60385), LmmCRK3 (Accession # AJ001275), LmmCRK4 (AJ293288),
LmajCRK3 (Accession # AF073381) TbCRK1 (Accession # X64314), TbCRK2
(Accession # X74598), TbCRK3 (Accession # X74617), TbCRK4 (AJ413200), TbCRK5
(Similar to Accession # NP_036103; Mus musculus MAPK, and Accession # NP_055041;
human MOK protein kinase), TbCRK6 (Similar to Accession # P54666; T. brucei Cell
Division Control Protein 2 homolog 3).

subfamily. However, they are more divergent from the CDC28.

latter that includes both TbCRK3 and TzCRK3, which is
in consistence with the fact that a minor evolutionary
difference does exist between Leishmania and Analysis of the structure
Trypanosoma. Moreover, there is almost no divergence
among the three leishmanial CRK3s, although LdCRK3 Analysis of the predicted amino acid sequence of the L.
has a single and two amino acids difference from donovani CRK3 has revealed a protein of 311 amino
LmCRK3 and LmajCRK3, respectively. This observation acids, which has an estimated molecular mass of 35.5
is also inconsistent with the fact that they are kDa. The isoelectric point (pI) is 6.8. The composition of
taxonomically from different complex, which give strong the amino acids has revealed that hydrophobic residues
support that CRK3 must be an essential kinase for present as 36.98%. L. donovani CRK3 also is rich in
Leishmania parasite. In spite of the fact that the leucine residues (12.54%), which may contribute largely
evolutionary rates of the yeast, Leishmania, to the hydrophobicity of the protein. Using the on-line
Trypanosoma, and human CDK family members were software (http://bioinformatics.weizmann.ac.il/hyd-
expected to be different, the CRK3 subfamily falls in the bin/plot_hydroph.pl) the hydropathicity of the L. donovani
same group with both human CDC2 and budding yeast CRK3 was compared with the human CDC2 (Figure 3).
220 Int. Res. J. Pharm. Pharmacol.

Figure 3. Comparison of the Hydropathic profile of L. donovani CRK3 & Human CDC2.
Using the on-line software, protein hydrophilicity/hydrophobicity search and comparison server, the hydropathicity
of the L. donovani CRK3 was compared with that of the human CDC2. Kyte Doolittle calculation method was used,
in which A= -1.8; C= -2.5; D, E, N, & Q= 3.5; F= -2.8; G= 0.4; H= 3.2; I= -4.5; K=3.9; L= -3.8; M= -1.9; P= 1.6; R=
4.5; S=0.8; T= 0.7; V= -4.2; W= 0.9; & Y= 1.3. Unlike, human CDC2, L. donovani CRK3 has a hydrophobic N-
terminal extension.

L. donovani CRK3 has a 19 aa extension on the N- active site of the enzyme-the region in which most of the
terminus, this analysis shows that it is hydrophobic. conserved residues lie (ATP-binding domain, PSTAIRE-
However, the two plots have more or less same box, and the T-loop). The larger amino-terminal lobe is
hydrophilicity/hydrophobicity pattern, particularly, in the composed of a seven-stranded antiparallel β-sheet. In
region 180-200 aa (human CDC2) and 200-220 aa (L. contrast, the smaller carboxy-terminal lobe is largely α-
donovani CRK3). helical, with two β-strands forming part of the cleft
between the lobes. The two phosphorylation sites that
correspond to the human CDC2 T-14 and Y-15 sites are
3D - Modelling located in the ATP-binding motif that is indicated by the
green arrow within β5 that lies near the cleft formed by
The availability of crystallographic models for members of the N-terminal and C-terminal domains. The C-terminal
the kinase family permits the use of structural domain is colored blue, with the activatory
information, along with primary sequence alignment, to phosphorylation site that corresponds to Thr-160 in the
model the structure of homologous enzymes. The overall human CDC2, highlighted in red in the T-loop which is
architecture of the catalytic core of the LdCKR3 is very illustrated in yellow, and lie near the αB.
similar to that of human CDK2, with a small and large From this model it is clear that the T-loop is protruding
lobe, in spite the fact that the overall amino acid identity in a similar way to that described for the inactive form of
is only 43%. In fact, the two kinases come from different all CDK protein kinases. Upon phosphorylation, this loop
branches of the kinase superfamily phylogenetic tree. will bend in a way to give the chance for the binding of
As shown in Figure 4 the molecule is illustrated in the cyclin partner, and thus bring the kinase to the active
forms of β-sheet shown as arrows and α-helix shown as status.
ribbons. All the α-helixes are shown in letters (A-J), while
the β-sheets are shown in numbers (1-9). The structure
of L. donovani CRK3 is that of a protein kinase catalytic Genomic organization of the L. donovani CRK3
core, consisting of an N-terminal domain formed
principally from seven β-sheets and four α-helixes, and a Southern blot analysis detected a single hybridising band
C-terminal domain formed principally from six α-helixes under high stringency conditions in five out of six
and two β-sheets. The kinase consists of two lobes with restriction digests (Figure 5, lanes 1-4, & 6). The SalI
a deep cleft between them, which may be represent the restriction digest (lane 5) gave one strong hybridising
 Ali et al. 221

(A)

(B)
Figure 4. Molecular modelling of L. donovani CRK3.
The three-dimensional homology modelling was performed using the Swiss-Pdb Viewer v3.6 b2.
The modelling was done using human cyclin-dependent kinase 2 (CDK2) complexed with the inhibitor
staurosporine as a template (accession code 1AQ1, Guex et al., 199921; and Guex and Peitsch, 199722). (A)
Using the successive colouring automatically, while [B] manual colouring, and the molecule is rotated to show
the important domains.
In (B), the N-terminal domain is principally white, with the exception of the 19 aa that shown by leucine 20
(colored yellow). The PQTALRE (corresponding to PSTAIRE in CDK2), is shown by the yellow helix and the
ATP-binding motif is illustrated in green arrow within β5. The C-terminal domain is colored blue with the
activation site (Thr-178) highlighted in red.
222 Int. Res. J. Pharm. Pharmacol.

1 2 3 4 5 6
kb
1 2 3 4 5 6
15.0
kb

15.0

6.0

6.0

3.3

3.3
Figure 5. Southern blot analysis of L. donovani genomic DNA, probed with the HindIII fragment of pGL89
under high stringency conditions.
6 µg of L. donovani strain (1s) (MHOM/SD/63/Khartoum) genomic DNA was digested with EcoRI (lane 1),
HindIII (lane 2), BglII (lane 3), SalI (lane 4), BamHI (lane 5), and NotI (lane 6), electrophoresed through a 0.8%
agarose gel, blotted to a nylon membrane and probed with the 2 kb HindIII fragment of pGL89. Arrowheads
indicate DNA markers in Kb.

fragment of approximately 3.3 kb and another weakly allow us to conclude that L. donovani CRK3 is essential,
hybridising fragment of approximately 6.0 kb. This result as there could be highly conserved non-essential
is consistent with the physical map created for the proteins. Rather it is likely to be essential because it has
putative L. donovani crk3 that showed an internal SalI been shown to be so in L. mexicana. Moreover, L.
site within the crk3 (data not shown). Together these donovani CRK3 is more similar to L. mexicana CRK3
results indicate that L. donovani crk3 is present as a than any other L. mexicana proteins such as L. mexicana
single copy gene in the L. donovani genome. tubulin (Accession # AF345947) or L. major actin-related
protein (Accession # AL445944) (data not shown), which
may indicate a similar function of both kinases in the two
DISCUSSION Leishmania species, possibly in the cell cycle control.
Interestingly, threonine and tyrosine residues known to
Full-length open reading frame (ORF) for the L. donovani be important in CDC2 regulation by phosphorylation are
CRK3 was obtained by screening a promastigote cDNA also conserved in L. donovani CRK3 (Thr-33 & Tyr-34).
library. Genomic southern analysis demonstrated that Therefore, the presence of these conserved residues in
the LdCRK3 is present as a single copy in the L. L. donovani CRK3 indicates that the Leishmania protein
donovani genome, which is consistent with the genomic kinase may be under similar post-translational control as
organization of the all so-far identified trypanosomatid CDC2-related kinases from other organisms.
CRKs. The putative protein encoded by L. donovani CRK3
Sequence comparison shows that all the has an N-terminal extension, which consists of 19-aa and
trypanosomatid cdc2-related kinases (CRKs) have more in this it is similar to other trypanosomatid CRK3 protein
or less the same degree of identity to either the human kinases. The role of this N-terminal extension is poorly
CDC2 or the budding yeast CDC28. This observation is understood, but plays no role in the regulation of the
consistent with what is expected from the divergence in kinase activity.
the evolution between the different eukaryotes; Phylogenetic analysis suggests that the CRK3s class
trypanosome parasite; yeast and human. Taken of protein kinases are good candidates to be the
together, these results suggest that the CRK3 homologue functional homologue of cdc2, as they fall in the same
is almost completely conserved and consequently is phylogenetic group with both the human CDC2 and the
essential in all the trypanosomatids, including T. brucei, budding yeast CDC28. However, it is not possible to use
T. cruzi, L. mexicana, L. major (Beverley et al., 1986), sequence identity to infer function. All CRKs are as
and L. donovani. However, the sequence data does not similar to human CDC2 as to each other and biochemical
 Ali et al. 223

or genetic data is required to determine a function. pharmacological studies on anti-leishmania agents have
T. brucei has an unusually high number of CRK genes been hampered by the insensitivity of Leishmania to
for a unicellular organism and this may indicate that the commonly used drugs. Therefore, the existence of
cell cycle regulatory mechanisms are so complicated that treatment failure highlighted the need to conduct studies
several molecules are required to co-ordinate the cell to investigate an appropriate anti-leishmanial agent. This
cycle steps during the complex life cycle. Early studies study have given the strong evidence that the CRK3
identified only three CRKs genes from Leishmania spp. class of Leishmania species is conserved and hence,
as compared to Trypanosoma. However, all these three have contributed to the current research on the
leishmanial CRKs have shown high structural and/or identification of the parasitic cdc2-related protein kinases
functional homology to the corresponding Trypanosome as drug targets from several protozoa; including
CRKs. Most recently five more Leishmanial CRKs (2, 4, Leishmania, African Trypanosoma, and Plasmodium
6, 7 and 8) were investigated in a search for their cyclin- falciparum.
partner and biological activity (Gomes et al., 2010). A Therefore, the possibility of designing a drug that can
similar large numbers of CRKs, could possibly be present be used in a costless manner to treat all forms of
in Leishmania, thus more genes are waiting to be leishmaniasis could be extrapolated.
identified in order to understand the whole picture of the
Leishmania cell cycle. For example a novel cyclin was
identified from L. mexicana (Ali et al., 2010) and was ACKNOWLEDGEMENTS
found to have ability to bind and activate L. mexicana
CRK3 (Gomes et al ., 2010). In a search for new drug We are very much grateful to Karen Grant, Paul Hassan,
against Leishmaniasis the active CRK3:CYCA complex and Philomena McCready (WCMP Glasgow) for their
was tried against known CDK inhibitors (Gomes et al., assistance. This work was supported by the UNDP/World
2010) and inhibitors against Leishmania CRK3-CYC6 Bank/WHO Special Programme for Research and
complex was identified most recently (Cleghorn et al., Training in Tropical Diseases (TDR) (grant ID. 971080).
2011).
Investigation of expression of L. donovani CRK3 gene
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