Phosphorylation-Dependent Assembly of a 14-3-3 Mediated Signaling Complex during Red Blood Cell Invasion by Plasmodium falciparum Merozoites

Abstract

Red blood cell (RBC) invasion by Plasmodium merozoites requires multiple steps that are regulated by signaling pathways. Exposure of P. falciparum merozoites to the physiological signal of low K+, as found in blood plasma, leads to a rise in cytosolic Ca2+, which mediates microneme secretion, motility, and invasion. We have used global phosphoproteomic analysis of merozoites to identify signaling pathways that are activated during invasion. Using quantitative phosphoproteomics, we found 394 protein phosphorylation site changes in merozoites subjected to different ionic environments (high K+/low K+), 143 of which were Ca2+ dependent. These included a number of signaling proteins such as catalytic and regulatory subunits of protein kinase A (PfPKAc and PfPKAr) and calcium-dependent protein kinase 1 (PfCDPK1). Proteins of the 14-3-3 family interact with phosphorylated target proteins to assemble signaling complexes. Here, using coimmunoprecipitation and gel filtration chromatography, we demonstrate that Pf14-3-3I binds phosphorylated PfPKAr and PfCDPK1 to mediate the assembly of a multiprotein complex in P. falciparum merozoites. A phospho-peptide, P1, based on the Ca2+-dependent phosphosites of PKAr, binds Pf14-3-3I and disrupts assembly of the Pf14-3-3I-mediated multiprotein complex. Disruption of the multiprotein complex with P1 inhibits microneme secretion and RBC invasion. This study thus identifies a novel signaling complex that plays a key role in merozoite invasion of RBCs. Disruption of this signaling complex could serve as a novel approach to inhibit blood-stage growth of malaria parasites. IMPORTANCE Invasion of red blood cells (RBCs) by Plasmodium falciparum merozoites is a complex process that is regulated by intricate signaling pathways. Here, we used phosphoproteomic profiling to identify the key proteins involved in signaling events during invasion. We found changes in the phosphorylation of various merozoite proteins, including multiple kinases previously implicated in the process of invasion. We also found that a phosphorylation-dependent multiprotein complex including signaling kinases assembles during the process of invasion. Disruption of this multiprotein complex impairs merozoite invasion of RBCs, providing a novel approach for the development of inhibitors to block the growth of blood-stage malaria parasites.

cAMP-Dependent Signaling Pathways as Potential Targets for Inhibition of Plasmodium falciparum Blood Stages

“…Putative PKA therapeutic targets can be identified by establishing protein kinase -protein substrate relationships from large scale protein phosphorylation data sets. Recent advances in liquid chromatography-tandem mass spectrometry have enabled large scale phosphoproteome screening of various P. falciparum life cycle stages, which has resulted in the detection of around 27,200 unique phosphorylation sites in proteins of intracellular asexual blood stage parasites (Solyakov et al, 2011;Treeck et al, 2011;Lasonder et al, 2012;Pease et al, 2013Pease et al, , 2018Collins et al, 2014;Alam et al, 2015;Kumar et al, 2017;Flueck et al, 2019;Patel et al, 2019) and 2,250 unique phosphorylation sites in proteins of extracellular merozoites (Lasonder et al, 2015;More et al, 2020). Patel et al (2019) studied directly the PKA-substrate relationship during the asexual erythrocytic cycle by a quantitative phosphoproteome analysis, comparing wild type parasites with transgenic parasites in which genes encoding PfPKAc and PfACβ (that synthesizes cAMP) were deleted using an inducible recombinase system.…”

“…The majority (46/61) of the phosphorylation sites contained the broad P. falciparum PKA consensus phosphorylation motif (Flueck et al, 2019), which included one or more basic residues at positions −2, −3, and −4 relative to the phosphorylated amino acid (Supplementary Table 1). More et al (2020) studied differences in P. falciparum protein phosphorylation levels in intra-erythrocytic merozoites prior to egress from the infected cell versus extracellular merozoites competent for red blood cell invasion. Switching merozoites from IC to EC conditions leads to a surge in cAMP concentration and activation of PfPKA, promoting merozoite invasion of RBCs (Dawn et al, 2014).…”

“…The list of 109 putative PfPKA substrate proteins enabled exploration of putative PfPKA-mediated signaling networks FIGURE 2 | Sequence logos of potential PKA substrate phosphorylation sites. Sequence logos of phosphorylation sites with a basic residue at position -3, extracted from 12 Plasmodium falciparum phosphoproteome studies (Treeck et al, 2011;Lasonder et al, 2012Lasonder et al, , 2015Pease et al, 2013Pease et al, , 2018Collins et al, 2014;Alam et al, 2015;Kumar et al, 2017;Flueck et al, 2019;Patel et al, 2019;More et al, 2020), were generated with Web Logo 3 (Crooks et al, 2004) and divided into the acidic, basic and neutral PfPKA motifs and the proline-directed non-PKA motif.…”

“…A rise in cytosolic levels of cAMP and Ca 2+ in merozoites suspended in a low K + ionic environment that mimics extracellular (EC) ionic conditions, activates PfPKA and PfCDPK1, respectively, which play essential roles in RBC invasion. Interestingly, quantitative phosphoproteomics of merozoites suspended in EC buffer, or in buffer mimicking intracellular (IC) ionic conditions with high K + , showed increased phosphorylation of both PfPKAr and PfCDPK1 in EC buffer compared to IC buffer ( More et al, 2020 ). The scaffold protein, 14-3-3, is known to interact with phosphorylated residues of target proteins to assemble protein complexes ( Agarwal-Mawal et al, 2003 ; Kawamoto et al, 2015 ).…”

“…The scaffold protein, 14-3-3, is known to interact with phosphorylated residues of target proteins to assemble protein complexes ( Agarwal-Mawal et al, 2003 ; Kawamoto et al, 2015 ). Antibodies against Pf14-3-3I (GeneID: PF3D7_0818200) immunoprecipitated Pf14-3-3, PfPKAr and PfCDPK1 from lysates of merozoites suspended in EC buffer suggesting that Pf14-3-3I and phosphorylated PfPKAr and PfCDPK1 form a multi-protein signaling complex ( More et al, 2020 ). In other eukaryotic cells, the subcellular spatio-temporal control of PKAc activity is maintained by interaction with AKAPs.…”