casein-kinase-ii and Malaria--Falciparum

Previous studies in model eukaryotes have demonstrated that phosphorylation of heterochromatin protein 1 (HP1) is important for dynamically regulating its various functions. However, in the malaria parasite Plasmodium falciparum both the function of HP1 phosphorylation and the identity of the protein kinases targeting HP1 are still elusive. In order to functionally analyze phosphorylation of P. falciparum HP1 (PfHP1), we first mapped PfHP1 phosphorylation sites by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of native PfHP1, which identified motifs from which potential kinases could be predicted; in particular, several phosphorylated residues were embedded in motifs rich in acidic residues, reminiscent of targets for P. falciparum casein kinase 2 (PfCK2). Secondly, we tested recombinant PfCK2 and a number of additional protein kinases for their ability to phosphorylate PfHP1 in in vitro kinase assays. These experiments validated our prediction that PfHP1 acts as a substrate for PfCK2. Furthermore, LC-MS/MS analysis showed that PfCK2 phosphorylates three clustered serine residues in an acidic motif within the central hinge region of PfHP1. To study the role of PfHP1 phosphorylation in live parasites we used CRISPR/Cas9-mediated genome editing to generate a number of conditional PfHP1 phosphomutants based on the DiCre/LoxP system. Our studies revealed that neither PfCK2-dependent phosphorylation of PfHP1, nor phosphorylation of the hinge domain in general, affect PfHP1's ability to localize to heterochromatin, and that PfHP1 phosphorylation in this region is dispensable for the proliferation of P. falciparum blood stage parasites.

Topics: Amino Acid Sequence; Casein Kinase II; Chromobox Protein Homolog 5; Chromosomal Proteins, Non-Histone; Heterochromatin; Humans; Malaria, Falciparum; Mutation; Phosphorylation; Plasmodium falciparum; Protozoan Proteins

Protein kinase CK2 (casein kinase 2) is a eukaryotic serine/threonine protein kinase with multiple substrates and roles in diverse cellular processes, including differentiation, proliferation, and translation. The mammalian holoenzyme consists of two catalytic alpha or alpha' subunits and two regulatory beta subunits. We report the identification and characterization of a Plasmodium falciparum CK2alpha orthologue, PfCK2alpha, and two PfCK2beta orthologues, PfCK2beta1 and PfCK2beta2. Recombinant PfCK2alpha possesses protein kinase activity, exhibits similar substrate and cosubstrate preferences to those of CK2alpha subunits from other organisms, and interacts with both of the PfCK2beta subunits in vitro. Gene disruption experiments show that the presence of PfCK2alpha is crucial to asexual blood stage parasites and thereby validate the enzyme as a possible drug target. PfCK2alpha is amenable to inhibitor screening, and we report differential susceptibility between the human and P. falciparum CK2alpha enzymes to a small molecule inhibitor. Taken together, our data identify PfCK2alpha as a potential target for antimalarial chemotherapeutic intervention.

Topics: Amino Acid Sequence; Animals; Casein Kinase II; Humans; Kinetics; Malaria, Falciparum; Molecular Sequence Data; Plasmodium falciparum; Protein Binding; Protein Subunits; Sequence Homology, Amino Acid; Substrate Specificity

Plasmodium falciparum malaria is a major human health scourge and a key cause of mortality. Its pathogenicity partly results from the phenomenon of "cytoadherence" mediated by the PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) family. Extracellular domains of PfEMP1s are variable and bind various host endothelial receptors, whereas their cytoplasmic domains (VARCs) are relatively conserved. VARCs affix PfEMP1s in the human erythrocyte membrane by interacting with host cytoskeleton proteins and exported parasite proteins. Here, we provide in vitro and in vivo evidence for PfEMP1 phosphorylation (on VARC) and propose an important function for this modification. Specific inhibitors and enhancers have been used to identify erythrocytic casein kinase II (CKII) as the enzyme responsible for VARC modification activity. We have also delineated probable CKII target residues on VARC, which mainly reside in an N-terminal acidic cluster. Our data show that VARC phosphorylation alters its binding to parasite encoded knob-associated histidine-rich protein (KAHRP). Finally, we demonstrate reduced cytoadherence of infected RBCs to endothelial receptors like ICAM-1 and CSA (these contribute to cerebral and placental malaria, respectively) in response to their CKII inhibition. Collectively, this study furthers our understanding of VARC function, underscores the importance of erythrocytic CKII in cytoadherence, and suggests a possible new target for anti-cytoadherence molecules.

Topics: Animals; Casein Kinase II; Cell Adhesion; Erythrocytes; Humans; Intercellular Adhesion Molecule-1; Malaria, Falciparum; Phosphorylation; Plasmodium falciparum; Protein Structure, Tertiary; Protozoan Proteins