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...