Specific phosphorylation of the PfRh2b invasion ligand of Plasmodium falciparum

Abstract

Red blood cell invasion by the malaria parasite Plasmodium falciparum relies on a complex protein network that uses low and high affinity receptor–ligand interactions. Signal transduction through the action of specific kinases is a control mechanism for the orchestration of this process. In the present study we report on the phosphorylation of the CPD (cytoplasmic domain) of P. falciparum Rh2b (reticulocyte homologue protein 2b). First, we identified Ser3233 as the sole phospho-acceptor site in the CPD for in vitro phosphorylation by parasite extract. We provide several lines of evidence that this phosphorylation is mediated by PfCK2 (P. falciparum casein kinase 2): phosphorylation is cAMP independent, utilizes ATP as well as GTP as phosphate donors, is inhibited by heparin and tetrabromocinnamic acid, and is mediated by purified PfCK2. We raised a phospho-specific antibody and showed that Ser3233 phosphorylation occurs in the parasite prior to host cell egress. We analysed the spatiotemporal aspects of this phosphorylation using immunoprecipitated endogenous Rh2b and minigenes expressing the CPD either at the plasma or rhoptry membrane. Phosphorylation of Rh2b is not spatially restricted to either the plasma or rhoptry membrane and most probably occurs before Rh2b is translocated from the rhoptry neck to the plasma membrane.

Parasite Calcineurin Regulates Host Cell Recognition and Attachment by Apicomplexans

“…Previously, we found that the cytoplasmic domain (CD) of the invasion ligand PfRh2b is physiologically phosphorylated in schizont-stage parasites (Engelberg et al, 2013). In vitro, extract from mature schizont-stage parasites displays calcium-dependent phosphatase activity toward phosphorylated recombinant PfRh2b-CD, perhaps consistent with dephosphorylation by calcineurin (Figure S4C).…”

The mechanics of malaria parasite invasion of the human erythrocyte – towards a reassessment of the host cell contribution

“…However, given Rh5's markedly reduced size and that it has proven to be essential to all parasite lines tested (others Rh's appear to show some redundancy), it is not clear whether it should be considered as an independent functional class of invasion protein (Weiss et al, 2015). Binding of Rh proteins has been implicated in intracellular signalling within the parasite, possibly governing further release of key effectors of invasion (Engelberg et al, 2013;Gao et al, 2013). Whether these proteins play a mechanistic role in the physical process of invasion is, however, entirely unknown.…”

PfRH2b specific monoclonal antibodies inhibit merozoite invasion

“…It has been shown that the cytoplasmic tail of PfRH2b is critical for its function (DeSimone et al ., ) and its phosphorylation by PfCK2 is important for signalling function. In addition, the cytoplasmic tail of PfRH2b may also function to control the recruitment of interacting partners necessary for regulating the process of invasion and other downstream signalling events (Engelberg et al ., ). In line with this, we have provided strong evidence that blocking of PfRH2b function inhibits merozoite Ca 2+ signalling thereby affecting the secretion of micronemal protein, EBA175, to the surface (Fig.…”

“…Mechanistically, the merozoite must engage with RBCs by reversible receptor interactions for its successful invasion. Upon active engagement resulting from the re-orientation of its apical end to the membrane, signals in the merozoite directs phosphorylation events at the cytoplasmic tail of PfRH2b (Engelberg et al, 2013). These signals direct change in the PfRH2b ecto-domain allowing an interaction with the RBC surface receptors.…”

“…It has been shown that the differential ability to use alternative invasion pathways is conferred by the cytoplasmic domains of PfRH2a and PfRH2b, not the ectodomain or transmembrane regions (Dvorin et al, 2010). PfRH2b at the cytoplasmic domain has been shown to be phosphorylated by P. falciparum casein kinase 2 (PfCK2) (Engelberg et al, 2013) and interact with aldolase (Pal-Bhowmick et al, 2012). Attempts to obtain a cytoplasmic-tailless PfRH2b proved that for the protein to be fully functional the cytoplasmic tail must be intact (Dvorin et al, 2010).…”