13 research outputs found
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
Validation of MS results by WB and proximity study by confocal microscopy for selected proteins.
<p>(A) Protein complexes of LFA-1, thrombospondin-1, talin-1, CD13 (top) and galectin-3 (bottom) in imDCs (day6) were co-immunoprecipitated with LFA-1. LFA-1 was enriched using mAb (clone SPV-L7) directed against αL (CD11a). mIgG1 coated beads were included as control IP. PNS: post nuclear supernatant. Samples were analysed in non-reducing conditions. (B) Confocal microscopy analysis of co-capping of LFA-1 and galectin-3, CD44 and CD71 on imDCs (day6). Receptor co-capping and staining were performed as described in <i>Material and Methods</i>. Antibodies against LFA-1 (clones: NKI-L15 and TS2/4) and CD71 are positive and negative markers for co-localization, respectively. Results are representatives of multiple cells per condition (n>10.) in two independent experiments. (C) To quantify the degree of co-localization between LFA-1 and binding candidates, Pearson´s coefficient was calculated. The values can vary between 0 and 1 (1 = 100% colocalization). P-values were compared to co-capping of LFA-1 with CD71 by two-tailed t-test, *** <0.001. Co-capping and staining were performed as described in Materials and Methods.</p
Protein-protein interaction of directly interacting LFA-1 binding candidates derived from DCs in stringent lysis conditions.
<p>A network was generated by uploading the protein names to the database of functional protein interactions (STRING v9.05) and retrieving experimentally proven direct protein-protein interactions. The resulting network was drawn by the authors. Based on our MS data, we could retrieve a high confidence network (score 0.6) of 19 directly interacting nodes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149637#pone.0149637.g005" target="_blank">Fig 5</a>), enriched in 31 connections. * 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