Identification, Cloning, and Mutational Analysis of the Casein Kinase 1 cDNA of the Malaria Parasite, Plasmodium falciparum

Abstract

The cDNA for casein kinase 1 (CK1) of Plasmodium falciparum was cloned, sequenced, and expressed in bacteria. The single major open reading frame of the 1.2-kilobase pair cDNA coded for a 324-amino acid polypeptide of ϳ37 kDa, the predicted sequence of which showed strong identity with known CK1 isoforms. The purified recombinant enzyme exhibited properties characteristic of CK1, such as inhibition by CK1-7, the ability to phosphorylate a highly specific peptide substrate, and a strong preference for ATP over GTP. A casein kinase activity, partially purified from soluble extracts of P. falciparum by affinity chromatography through CK1-7 columns displayed identical properties. The activity showed a stage-specific expression in the parasite, in the order trophozoite > ring > > schizont. Northern analysis indicated the existence of two major CK1 mRNAs, 2.4 and 3.2 kilobase pairs long, the levels of which were in the order ring > schizont > trophozoite. Mutagenesis of recombinant CK1 defined important amino acid residues and their potential role in the conformation of the enzyme. The malarial CK1 appeared to be the one of the smallest and perhaps the most primitive CK1 enzymes known, containing little sequence information beyond the minimal catalytic domain.The parasitic protozoan, Plasmodium falciparum, is the causative agent of malaria throughout the world and is responsible for an annual death toll of nearly 3 million, the majority of which are children and pregnant mothers (1). However, tools available to control malaria are inadequate, and drug-resistant strains are widespread; moreover, the immediate prospect of a useful vaccine is uncertain. Thus, there is an urgent need to obtain fundamental knowledge about the various cellular processes of P. falciparum at the molecular level, so that susceptible targets can be identified. With this long-term goal in mind, we have initiated studies of the signal transduction system in P. falciparum. Since reversible protein phosphorylation and dephosphorylation constitute a major mechanism of signal transduction (2), one of our immediate goals has been to characterize the various protein kinases in P. falciparum. In this paper, we report the characterization, expression, stagespecific regulation, and mutational analysis of P. falciparum casein kinase 1 (PfCK1). 1 Casein kinase-1 and -2 (CK1 and CK2) are multipotential Ser/Thr protein kinases, originally purified from rabbit reticulocyte lysates using casein as substrate (reviewed in Refs. 3 and 4). In the subsequent years, both enzymes were shown to phosphorylate, and thus regulate, a wide variety of cellular proteins. The sequence alignment of CK1 genes of various organisms and deletion analysis of recombinant yeast CK1 have recently resulted in the delineation of the following domains in the prototype 45-kDa yeast enzyme (summarized in Refs. 5 and 6): an N-terminal catalytic domain of about 300 amino acids followed by a 12-residue stretch conserved among some forms but not in others; a hydrophilic 85-residue segment predi...

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“…Total RNA was isolated from asynchronous P. falciparum 3D7 cells grown in human A-positive erythrocytes essentially as before [48,49]. Various pairs of primers were designed on the basis of the relevant sequences of chromosome 14.…”

“…Growth and induction of E. coli BL21(DE3) containing pET-15b-PfPP1 and the RIG plasmid were carried out using procedures described earlier [14,48], except that the culture was grown at 18°C in the presence of 2 mM MnCl 2 , and IPTG concentration was lowered to 0.4 mM. The (His) 6 -tagged PfPP1 expressed from pET-15b-PfPP1 was purified through Ni +2 -chelation chromatography [14] as described by the manufacturer (Novagen), with 1 mM MnCl 2 being present in all the buffers.…”

Regulation of protein phosphatase type 1 and cell cycle progression by PfLRR1, a novel leucine‐rich repeat protein of the human malaria parasite Plasmodium falciparum

“…It is well established that phosphorylation/dephosphorylation of proteins is an important mechanism in the control of various events in cells (Cohen, 1989;Luan, 2003). Efforts have begun for an extensive examination of the potential components involved in signalling pathways in P. falciparum , comprising protein kinase and phosphatase genes (Barik et al ., 1997;Doerig et al ., 2002;Ward et al ., 2004). Enzymatic activities and genes related to plasmodial phosphatases (PPs) have been identified.…”

Pharmacological Assessment Defines Leishmania donovani Casein Kinase 1 as a Drug Target and Reveals Important Functions in Parasite Viability and Intracellular Infection

“…Members of another family of kinases have emerged as potential drug targets, casein kinase 1 (CK1), a family of multifunctional Ser/Thr protein kinases that are characterized by a highly conserved kinase domain and a specific C-terminal domain implicated in its regulation and localization (8). Although CK1 homologs in various protozoa share properties that are similar to those of higher eukaryotes, their functions have not yet been identified (9)(10)(11)(12)(13)(14). There are six CK1 isoforms in Leishmania major, LmjF35.1000 (UniProtKB/Swiss-Prot accession number Q9NHE2; LmaCK1.1), LmjF35.1010 (UniProtKB/Swiss-Prot accession number Q9NHE1; LmaCK1.2), LmjF04.1210 (UniProtKB/ Swiss-Prot accession number O96999), LmjF25.1580 (UniProtKB/ Swiss-Prot accession number Q4Q9T3), LmjF27.1780 (GenBank accession number E9ADF5), and LmjF30.3470 (UniProtKB/ Swiss-Prot accession number Q4Q6U1), for which the biochemical properties and the physiological functions are largely unknown.…”