Abstract
Casein kinase 1 (CK1) is a pleiotropic protein kinase implicated in several fundamental processes of eukaryotic cell biology. Plasmodium falciparum encodes a single CK1 isoform, PfCK1, that is expressed at all stages of the parasite’s life cycle. We have previously shown that the pfck1 gene cannot be disrupted, but that the locus can be modified if no loss-of-function is incurred, suggesting an important role for this kinase in intra-erythrocytic asexual proliferation. Here, we report on the use of parasite lines expressing GFP- or His-tagged PfCK1 from the endogenous locus to investigate (i) the dynamics of PfCK1 localisation during the asexual cycle in red blood cells, and (ii) potential interactors of PfCK1, so as to gain insight into the involvement of the enzyme in specific cellular processes. Immunofluorescence analysis reveals a dynamic localisation of PfCK1, with evidence for a pool of the enzyme being directed to the membrane of the host erythrocyte in the early stages of infection, followed by a predominantly intra-parasite localisation in trophozoites and schizonts and association with micronemes in merozoites. Furthermore, we present strong evidence that a pool of enzymatically active PfCK1 is secreted into the culture supernatant, demonstrating that PfCK1 is an ectokinase. Our interactome experiments and ensuing kinase assays using recombinant PfCK1 to phosphorylate putative interactors in vitro suggest an involvement of PfCK1 in many cellular processes such as mRNA splicing, protein trafficking, ribosomal, and host cell invasion.
Highlights
Human malaria is caused by infection with protozoan parasites of the genus Plasmodium, and P. falciparum, the most virulent species, is responsible for the vast majority of lethal cases. 40% of the world’s population is at risk of contracting the disease, and 300 million clinical cases as well as 650,000 deaths are reported yearly, mostly among young children in sub-Saharan Africa
The expression pattern of PfCK1 during the asexual erythrocytic cycle of wild-type parasites was investigated by immunoblot and immunofluorescence analyses using a rabbit antibody directed against a PfCK1-derived synthetic peptide
We showed in the context of our kinome-wide reverse genetics study [6] that the pfck1 gene is refractory to disruption, but can be modified by His4 C-terminal tagging, indicating that PfCK1 function is likely essential for parasite proliferation in erythrocytes
Summary
Human malaria is caused by infection with protozoan parasites of the genus Plasmodium, and P. falciparum, the most virulent species, is responsible for the vast majority of lethal cases. 40% of the world’s population is at risk of contracting the disease, and 300 million clinical cases as well as 650,000 deaths are reported yearly, mostly among young children in sub-Saharan Africa. Human malaria is caused by infection with protozoan parasites of the genus Plasmodium, and P. falciparum, the most virulent species, is responsible for the vast majority of lethal cases. Together with the fact that gene expression in malaria parasites is to a large extent regulated by post-translational mechanisms, this suggests that protein phosphorylation is an important feature in these organisms [4]. This is corroborated by the large number of phosphoproteins detected by mass spectrometry analyses [5,6,7,8]. In view of the success of targeting protein kinases in cancer chemotherapy, illustrated by the approval of several kinase inhibitors as anticancer drugs over the recent years [9], the Plasmodium kinome has been highlighted as a potential target for antimalarials with novel modes of action [10,11]
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