Plasmodium falciparum raf kinase inhibitor is a lipid binding protein that interacts with and regulates the activity of PfCDPK1, an essential plant-like kinase required for red blood cell invasion

Abstract

Raf Kinase Inhibitor Protein (RKIP) is an important regulator of the MAPK signaling pathway in multicellular eukaryotes. Plasmodium falciparum RKIP (PfRKIP) is a putative phosphatidylethanolamine binding protein (PEBP) that shares limited similarity with Homo sapiens RKIP (HsRKIP). Interestingly, critical components of the MAPK pathway are not expressed in malaria parasites and the physiological function of PfRKIP remains unknown. PfRKIP is expressed throughout the asexual schizogony with maximum expression in late schizonts. Interestingly, PfRKIP and HsRKIP show pH-dependent differential interaction profiles with various lipids. At physiological pH, PfRKIP shows interaction with phosphatidic acid and lipids containing phosphorylated phosphatidylinositol group; however, HsRKIP shows no interaction under the same conditions. Mutation of conserved residues in the PEBP domain of PfRKIP decreases its interaction with PtdIns(3)P. Additionally, in silico docking and mutagenesis studies identified a unique IKK motif within the PEBP domain of PfRKIP that is important for its interaction with the lipids. Using ELISA, we demonstrate the interaction of PfRKIP with PfCDPK1. Importantly, we establish the interaction of PfRKIP and PfCDPK1 within the parasites using immunofluorescence assay and proximity biotinylation technique. Furthermore, our results suggest that PfRKIP regulates the kinase activity of PfCDPK1. In the presence of its substrate, PfCDPK1 hyper-phosphorylates PfRKIP which leads to its dissociation from PfCDPK1. Dissociation of PfRKIP allows PfCDPK1 to trans-phosphorylate its substrates. The molecular mechanism of interaction between PfRKIP and PfCDPK1 may be explored further to identify novel anti-malarial compounds.