Phosphorylation-Dependent Assembly of a 14-3-3 Mediated Signaling Complex During Red Blood Cell Invasion byPlasmodium falciparumMerozoites

Abstract

232 words; Text: 5494 words 23 24 25 Abstract 29Red blood cell (RBC) invasion by Plasmodium merozoites requires multiple steps that are regulated by 30 signaling pathways. Exposure of P. falciparum merozoites to the physiological signal of low K + , as 31 found in blood plasma, leads to a rise in cytosolic Ca 2+ , which mediates microneme secretion, motility, 32 and invasion. We have used global phosphoproteomic analysis of merozoites to identify signaling 33 pathways that are activated during invasion. Using quantitative phosphoproteomics we found 394 34 protein phosphorylation site changes in merozoites subjected to different ionic environments 35 (high K + / low K + ) out of which 143 were Ca 2+ -dependent. These included a number of signaling 36 proteins such as catalytic and regulatory subunits of protein kinase A (PfPKAc and PfPKAr) and 37 calcium-dependent protein kinase 1 (PfCDPK1). Proteins of the 14-3-3 family interact with 38 phosphorylated target proteins to assemble signaling complexes. Here, using co-immunoprecipitation 39 and gel filtration chromatography, we demonstrate that Pf14-3-3I binds phosphorylated PfPKAr and 40 PfCDPK1 to mediate the assembly of a multi-protein complex in P. falciparum merozoites. A phospho-41 peptide, P1, based on the Ca 2+ dependent phosphosites of PKAr, binds Pf14-3-3I and disrupts assembly 42 of the Pf14-3-3I-mediated multi-protein complex. Disruption of the multi-protein complex with P1 43 inhibits microneme secretion and RBC invasion. This study thus identifies a novel signaling complex 44 that plays a key role in merozoite invasion of RBCs. Disruption of this signaling complex could serve 45 as a novel approach to inhibit blood stage growth of malaria parasites. 46 47 48Invasion of red blood cells (RBCs) by Plasmodium falciparum merozoites is a complex process that is 49 regulated by intricate signaling pathways. Here, we have used phosphoproteomic profiling to identify 50 the key proteins involved in signaling events during invasion. We found changes in the 51 phosphorylation of various merozoite proteins including multiple kinases previously implicated in the 52 process of invasion. We also found that a phosphorylation dependent multi-protein complex including 53 signaling kinases assembles during the process of invasion. Disruption of this multi-protein complex 54 impairs merozoite invasion of RBCs providing a novel approach for the development of inhibitors to 55 block the growth of blood stage malaria parasites. 56 57 58 59 60 the parasite life cycle during which merozoites invade and multiply within host red blood cells (RBCs). 63 Following the development of mature schizonts, newly formed merozoites egress and invade 64 uninfected RBCs to initiate a new cycle of infection. The invasion of RBCs by P. falciparum 65 merozoites is a complex multi-step process that is mediated by specific molecular interactions between 66 red cell surface receptors and parasite protein ligands (1). These ligands are initially located in internal 67 secretory organelles ca...

CDPKs: The critical decoders of calcium signal at various stages of malaria parasite development

“…2 ) [50] . Post-translational modifications of CDPKs are responsible for modulating the activity, localization and interaction of CDPKs with their cognate substrates [84] , [79] , [2] . Myristoylation of a conserved glycine residue (Gly 2 ) at the N-terminus of PfCDPK1 is critical for its membrane localization in schizonts and merozoites [84] .…”

“…It has been suggested that phosphorylation of RhopH3 is mediated by CDPK1 [33] . A recent elegant study by More et al, demonstrates formation of a multimeric complex in merozoites involving phosphorylated CDPK1, regulatory subunit of PKA (PKAr) and Pf14-3-3I that plays a critical role in the invasion process and microneme discharge [79] . These studies suggest an essential role for CDPK1 in asexual proliferation of the parasite and as such, corroborates well with the inability to delete CDPK1 in the WT parasite background.…”

“…Paradoxically, PKG dependent S64 phosphorylation of CDPK1 that was also identified in vivo did not affect the kinase activity of CDPK1. Interestingly, preferential localization of phosphorylated CDPK1 at the apical end of merozoites is indicative that phosphorylation dependent spaciotemporal modulation and formation of higher molecular weight complexes is crucial for parasite invasion of RBCs [2] , [79] . Overall, molecular details of CDPK1 mediated apical organelle discharge and invasion are not very well understood and needs further investigation.…”

“… CDPKs Expression & Localisation Function in Plasmodium spp. References Putative Orthologous AtCDPKs* (percentage identity) P. falciparum CDPK1 Asexual (Rings, Trophozoites, Schizonts, Merozoites) Sexual (Gametocytes, Gametes) Merozoite invasion, Schizont development and egress Male and Female gametogenesis, Mosquito transmission [84] , [9] , [59] , [5] , [2] , [8] , [55] , [37] , [33] , [79] [6] AtCDPK29 (37%) /23 (36%) /21 (36%) /9 (36%) /26 (36%) CDPK2 Asexual (Trophozoites, Schizonts) Sexual (Gametocytes) - Male Gametocyte Exflagellation and Mosquito infection [7] [7] AtCDPK29 (40%) /30 (39%) /4 (39%) /9 (39%) /5 (39%) CDPK3 Sexual (Gametocytes) – [69] AtCDPK33 (41%) /29 (40%) /9 (40%) /4 (40%) CDPK4 - Sexual (Gametocytes) Merozoite Invasion Male Gametocyte Exflagellation and mosquito transmission [35] [58] , [97] , [112] , [91] , [90] AtCDPK29 (40%) /21 (39%)/ 26 (37%)/ 34 (37%) CDPK5 Asexual (Schizonts, Merozoites) Merozoite egress (Release of Egress-Specific Organelles) [32] [1] AtCDPK25 (39%) /1 (38%) /21 (37%) /29 (37%) /23 (37%) …”

Functional inactivation of Plasmodium falciparum glycogen synthase kinase GSK3 modulates erythrocyte invasion and blocks gametocyte maturation

“…Protein kinases possess a strong potential as antimalarial targets given their crucial role for parasite survival ( 5 ). A substantial body of work highlights the role of this enzyme class in the regulation of host cell invasion ( 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ). For instance, the physiological role of protein kinase A, casein kinase 2 (CK2) as well as glycogen synthase kinase 3 (GSK3) during the host cell invasion has been demonstrated ( 6 , 7 , 8 , 9 , 10 , 16 , 17 , 18 , 19 ).…”