The <i>Plasmodium falciparum</i> Schizont Phosphoproteome Reveals Extensive Phosphatidylinositol and cAMP-Protein Kinase A Signaling

The asexual blood stages of <i>Plasmodium falciparum</i> cause the most lethal form of human malaria. During growth within an infected red blood cell, parasite multiplication and formation of invasive merozoites is called schizogony. Here, we present a detailed analysis of the phosphoproteome of <i>P. falciparum</i> schizonts revealing 2541 unique phosphorylation sites, including 871 novel sites. Prominent roles for cAMP-dependent protein kinase A- and phosphatidylinositol-signaling were identified following analysis by functional enrichment, phosphoprotein interaction network clustering and phospho-motif identification tools. We observed that most key enzymes in the inositol pathway are phosphorylated, which strongly suggests additional levels of regulation and crosstalk with other protein kinases that coregulate different biological processes. A distinct pattern of phosphorylation of proteins involved in merozoite egress and red blood cell invasion was noted. The analyses also revealed that cAMP-PKA signaling is implicated in a wide variety of processes including motility. We verified this finding experimentally using an in vitro kinase assay and identified three novel PKA substrates associated with the glideosome motor complex: myosin A, GAP45 and CDPK1. Therefore, in addition to an established role for CDPK1 in the motor complex, this study reveals the coinvolvement of PKA, further implicating cAMP as an important regulator of host cell invasion

The <i>Plasmodium falciparum</i> Schizont Phosphoproteome Reveals Extensive Phosphatidylinositol and cAMP-Protein Kinase A Signaling

The asexual blood stages of <i>Plasmodium falciparum</i> cause the most lethal form of human malaria. During growth within an infected red blood cell, parasite multiplication and formation of invasive merozoites is called schizogony. Here, we present a detailed analysis of the phosphoproteome of <i>P. falciparum</i> schizonts revealing 2541 unique phosphorylation sites, including 871 novel sites. Prominent roles for cAMP-dependent protein kinase A- and phosphatidylinositol-signaling were identified following analysis by functional enrichment, phosphoprotein interaction network clustering and phospho-motif identification tools. We observed that most key enzymes in the inositol pathway are phosphorylated, which strongly suggests additional levels of regulation and crosstalk with other protein kinases that coregulate different biological processes. A distinct pattern of phosphorylation of proteins involved in merozoite egress and red blood cell invasion was noted. The analyses also revealed that cAMP-PKA signaling is implicated in a wide variety of processes including motility. We verified this finding experimentally using an in vitro kinase assay and identified three novel PKA substrates associated with the glideosome motor complex: myosin A, GAP45 and CDPK1. Therefore, in addition to an established role for CDPK1 in the motor complex, this study reveals the coinvolvement of PKA, further implicating cAMP as an important regulator of host cell invasion

The <i>Plasmodium falciparum</i> Schizont Phosphoproteome Reveals Extensive Phosphatidylinositol and cAMP-Protein Kinase A Signaling

The asexual blood stages of <i>Plasmodium falciparum</i> cause the most lethal form of human malaria. During growth within an infected red blood cell, parasite multiplication and formation of invasive merozoites is called schizogony. Here, we present a detailed analysis of the phosphoproteome of <i>P. falciparum</i> schizonts revealing 2541 unique phosphorylation sites, including 871 novel sites. Prominent roles for cAMP-dependent protein kinase A- and phosphatidylinositol-signaling were identified following analysis by functional enrichment, phosphoprotein interaction network clustering and phospho-motif identification tools. We observed that most key enzymes in the inositol pathway are phosphorylated, which strongly suggests additional levels of regulation and crosstalk with other protein kinases that coregulate different biological processes. A distinct pattern of phosphorylation of proteins involved in merozoite egress and red blood cell invasion was noted. The analyses also revealed that cAMP-PKA signaling is implicated in a wide variety of processes including motility. We verified this finding experimentally using an in vitro kinase assay and identified three novel PKA substrates associated with the glideosome motor complex: myosin A, GAP45 and CDPK1. Therefore, in addition to an established role for CDPK1 in the motor complex, this study reveals the coinvolvement of PKA, further implicating cAMP as an important regulator of host cell invasion

The <i>Plasmodium falciparum</i> Schizont Phosphoproteome Reveals Extensive Phosphatidylinositol and cAMP-Protein Kinase A Signaling

The asexual blood stages of <i>Plasmodium falciparum</i> cause the most lethal form of human malaria. During growth within an infected red blood cell, parasite multiplication and formation of invasive merozoites is called schizogony. Here, we present a detailed analysis of the phosphoproteome of <i>P. falciparum</i> schizonts revealing 2541 unique phosphorylation sites, including 871 novel sites. Prominent roles for cAMP-dependent protein kinase A- and phosphatidylinositol-signaling were identified following analysis by functional enrichment, phosphoprotein interaction network clustering and phospho-motif identification tools. We observed that most key enzymes in the inositol pathway are phosphorylated, which strongly suggests additional levels of regulation and crosstalk with other protein kinases that coregulate different biological processes. A distinct pattern of phosphorylation of proteins involved in merozoite egress and red blood cell invasion was noted. The analyses also revealed that cAMP-PKA signaling is implicated in a wide variety of processes including motility. We verified this finding experimentally using an in vitro kinase assay and identified three novel PKA substrates associated with the glideosome motor complex: myosin A, GAP45 and CDPK1. Therefore, in addition to an established role for CDPK1 in the motor complex, this study reveals the coinvolvement of PKA, further implicating cAMP as an important regulator of host cell invasion

The <i>Plasmodium falciparum</i> Schizont Phosphoproteome Reveals Extensive Phosphatidylinositol and cAMP-Protein Kinase A Signaling

The asexual blood stages of <i>Plasmodium falciparum</i> cause the most lethal form of human malaria. During growth within an infected red blood cell, parasite multiplication and formation of invasive merozoites is called schizogony. Here, we present a detailed analysis of the phosphoproteome of <i>P. falciparum</i> schizonts revealing 2541 unique phosphorylation sites, including 871 novel sites. Prominent roles for cAMP-dependent protein kinase A- and phosphatidylinositol-signaling were identified following analysis by functional enrichment, phosphoprotein interaction network clustering and phospho-motif identification tools. We observed that most key enzymes in the inositol pathway are phosphorylated, which strongly suggests additional levels of regulation and crosstalk with other protein kinases that coregulate different biological processes. A distinct pattern of phosphorylation of proteins involved in merozoite egress and red blood cell invasion was noted. The analyses also revealed that cAMP-PKA signaling is implicated in a wide variety of processes including motility. We verified this finding experimentally using an in vitro kinase assay and identified three novel PKA substrates associated with the glideosome motor complex: myosin A, GAP45 and CDPK1. Therefore, in addition to an established role for CDPK1 in the motor complex, this study reveals the coinvolvement of PKA, further implicating cAMP as an important regulator of host cell invasion

The <i>Plasmodium falciparum</i> Schizont Phosphoproteome Reveals Extensive Phosphatidylinositol and cAMP-Protein Kinase A Signaling

The asexual blood stages of <i>Plasmodium falciparum</i> cause the most lethal form of human malaria. During growth within an infected red blood cell, parasite multiplication and formation of invasive merozoites is called schizogony. Here, we present a detailed analysis of the phosphoproteome of <i>P. falciparum</i> schizonts revealing 2541 unique phosphorylation sites, including 871 novel sites. Prominent roles for cAMP-dependent protein kinase A- and phosphatidylinositol-signaling were identified following analysis by functional enrichment, phosphoprotein interaction network clustering and phospho-motif identification tools. We observed that most key enzymes in the inositol pathway are phosphorylated, which strongly suggests additional levels of regulation and crosstalk with other protein kinases that coregulate different biological processes. A distinct pattern of phosphorylation of proteins involved in merozoite egress and red blood cell invasion was noted. The analyses also revealed that cAMP-PKA signaling is implicated in a wide variety of processes including motility. We verified this finding experimentally using an in vitro kinase assay and identified three novel PKA substrates associated with the glideosome motor complex: myosin A, GAP45 and CDPK1. Therefore, in addition to an established role for CDPK1 in the motor complex, this study reveals the coinvolvement of PKA, further implicating cAMP as an important regulator of host cell invasion

The <i>Plasmodium falciparum</i> Schizont Phosphoproteome Reveals Extensive Phosphatidylinositol and cAMP-Protein Kinase A Signaling

The asexual blood stages of <i>Plasmodium falciparum</i> cause the most lethal form of human malaria. During growth within an infected red blood cell, parasite multiplication and formation of invasive merozoites is called schizogony. Here, we present a detailed analysis of the phosphoproteome of <i>P. falciparum</i> schizonts revealing 2541 unique phosphorylation sites, including 871 novel sites. Prominent roles for cAMP-dependent protein kinase A- and phosphatidylinositol-signaling were identified following analysis by functional enrichment, phosphoprotein interaction network clustering and phospho-motif identification tools. We observed that most key enzymes in the inositol pathway are phosphorylated, which strongly suggests additional levels of regulation and crosstalk with other protein kinases that coregulate different biological processes. A distinct pattern of phosphorylation of proteins involved in merozoite egress and red blood cell invasion was noted. The analyses also revealed that cAMP-PKA signaling is implicated in a wide variety of processes including motility. We verified this finding experimentally using an in vitro kinase assay and identified three novel PKA substrates associated with the glideosome motor complex: myosin A, GAP45 and CDPK1. Therefore, in addition to an established role for CDPK1 in the motor complex, this study reveals the coinvolvement of PKA, further implicating cAMP as an important regulator of host cell invasion

Protein-protein interaction network of directly interacting LFA-1 binding candidates derived from DCs.

<p>(A) Comparison of immunoprecipitated LFA-1 from day 6 imDCs in mild and stringent lysis conditions. Venn diagrams of proteins identified in DCs in stringent (red) and mild (blue) IP conditions. Numbers of identified proteins, as well as common proteins (yellow) are indicated. (B) A network of LFA-1 (heterodimer formed by an αL (ITGAL) and β2 (ITGB2) chain) binding partners was generated by fusing the data sets derived from mild and stringent IP conditions in DCs, uploading the protein names to the database of functional protein interactions (STRING v9.05) and retrieving experimental proven direct protein-protein interactions. (ribosomal and histone complexes were removed for better visualization of proteins involved in integrin function). The resulting network was redrawn by the authors. Based on our MS data, we could construct a high confidence network (score 0.6) containing 78 nodes and 154 connections. Blue nodes represent proteins identified in mild lysis conditions, and red nodes represent proteins identified in stringent lysis conditions. Green nodes represent proteins identified both in mild and stringent IP condition. * indicates an interaction that was not present in the STRING database (version 9.1) with experimental support, but this node and interactions were added by the authors based on the current literature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149637#pone.0149637.ref027" target="_blank">27</a>].</p

Ingenuity Pathway Analysis: Top Canonical Pathways in DCs (stringent IP conditions).

<p>Ingenuity Pathway Analysis: Top Canonical Pathways in DCs (stringent IP conditions).</p

Comparison of total LFA-1 binding partners (derived from mild and stringent lysis conditions) in monocytes and DCs.

<p>(A) Venn diagrams of proteins identified in monocytes (blue) and DCs (yellow). Numbers of identified proteins, as well as common proteins are indicated. (B) PPI network of directly interacting LFA-1 (heterodimer formed by and αL (ITGAL) and β2 (ITGB2) chain) binding partners derived from monocytes in stringent lysis and mild lysis conditions. A network was generated by uploading the protein names to the database of functional protein interactions (STRING v9.05) and retrieving experimental proven direct protein-protein interactions. The resulting network was drawn by the authors. Based on our MS data, we could retrieve 3 high confidence networks (score 0.6), with a maximum of 19 directly interconnected nodes. Blue nodes represent proteins identified in mild lysis conditions, and red nodes represent proteins identified in stringent lysis conditions.</p