Parasite morphology and global protein phosphorylation pattern of PKG inhibitor-treated <i>P. falciparum</i> schizonts.

<p>(<b>A</b>) Immunofluorescent staining of DMSO/compound 1-treated WT schizonts using antibodies detecting (i) PfGAP45 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Yeoman1" target="_blank">[34]</a>, (ii) PfSUB1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Yeoh1" target="_blank">[14]</a> and (iii) PfAMA1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Harris1" target="_blank">[23]</a>. Representative images are shown for each staining together with parasite nuclei stained with DAPI. Bars ∼5 µM. (<b>B</b>) Metabolic labelling of phosphoproteins in <i>P. falciparum</i> schizonts. Autoradiographs of (i) 3D7 WT and (ii) gatekeeper mutant 3D7 PfPKG_T618Q schizonts, treated with ³²P-orthophosphate and DMSO (−) or compound 2 (+) prior to lysis, ÄKTA anion exchange chromatography (fractions 10–14 are shown) and separation by SDS-PAGE. Rectangular boxes highlight bands that show a differential signal following inhibitor-treatment in WT, but not PfPKG_T618Q schizonts.</p

Subcellular location of PfPKG in mature schizonts.

<p>Dual immunofluorescent detection of PfPKG-HA in fixed smears of early and late schizonts of the PfPKG-HA-3A clone together with (<b>A</b>) PfGAPDH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Daubenberger1" target="_blank">[30]</a>, (<b>B</b>) PfBiP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-vanDooren1" target="_blank">[32]</a>, (<b>C</b>) PfPMV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Klemba1" target="_blank">[31]</a>, (<b>D</b>) PfRab11A <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-AgopNersesian1" target="_blank">[33]</a> and (<b>E</b>) PfGAP45 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Yeoman1" target="_blank">[34]</a>. Representative images are shown for each antibody, together with bright field images (first column) and parasite nuclei stained with DAPI (in the merged image). Bars ∼5 µM. To quantify co-localisation, Pearson coefficients <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Manders1" target="_blank">[36]</a> of the individual stains were calculated using Imaris image analysis software (Bitplane).</p

PfPKG expression peaks in late blood stages and is carbonate-soluble.

<p>(<b>A</b>) Western blots of synchronised cultures of the PfPKG-HA-3A clone and WT parasites (3D7 clone), 24 hours (mostly mid trophozoites), 30 hours (mostly late trophozoites), 41 hours (mostly early schizonts) and 46 hours (mostly late schizonts) post invasion were detected with anti-HA and anti-humanPKG, respectively. Blots were re-probed with an antibody against Pfαtubulin to estimate the relative total protein loading between lanes. (<b>B</b>) Sequential solubilisation of parasite proteins from saponin-released late trophozoites and schizonts. S1: soluble protein fraction (5 mM Tris-HCl, freeze thaw); S2: peripheral membrane fraction (extraction with 100 mM Na₂CO₃); S3: integral membrane fraction (extraction with 4% SDS/0.5% TX-114/0.5×PBS). Equal volumes of the three supernatants were analysed by SDS-PAGE and Western blots were probed for the integral membrane protein PfPMV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Klemba1" target="_blank">[31]</a>, stripped and re-probed simultaneously for PfGAPDH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Daubenberger1" target="_blank">[30]</a> and PfPKG-HA. Densitometric analysis of the scan of the blot presented revealed that 89.3% of PfPKG-HA is present in fraction S1, while fractions S2 and S3 contain 9.5% and 1.2%, respectively. (<b>C</b>) Immunofluorescent anti-HA detection in fixed smears of erythrocytic stages of the PfPKG-HA-3A clone. Representative images of (i) a ring stage parasite, (ii) three early trophozoites, (iii) an early schizont, (iv, v) late schizonts (approximate hours post invasion: (i) 4–10, (ii) 20–26, (iii) 33–39, (iv, v) 45–48) and (vi) a stage III gametocyte are shown together with bright field images (first column) and parasite nuclei stained with DAPI (second column). Bars ∼5 µM.</p

PfCK2 auto-phosphorylates <i>in vitro</i> on threonine 63.

<p><b>A</b>: <i>In vitro</i> kinase assay for GST-PfCK2 autophosphorylation, top panel: autoradiograph, bottom panel: Coomassie stain. <b>B</b>: LC-MS/MS trace identifying phosphorylation of PfCK2 at T63; right: Also shown is the hypothetical fragmentation table where the b-ions and y-ions detected in the LC-MS/MS spectra are shown in red and bold, respectively. <b>C</b>: Sequence of PfCK2 showing the phosphopeptide identified in the LC-MS/MS analysis (underlined) and the threonine 63 phosphorylation site (in red).</p

PfCK2 phosphorylates MCM2 on Ser13 and Tyr16 <i>in vitro</i>.

<p><b>A</b>: In vitro kinase assay using a GST fusion protein containing a N-terminal portion of MCM2 (GST-MCM2) or the same fusion protein but where residue Y16 is mutated to an phenylalanine (Y16F) or where residue S13 is mutated to an alanine (S13A) or where both S13 and Y16 are mutated to an alanine and phenylalanine respectively (S13A/Y16F). Top panel: autoradiograph, bottom panel: Coomassie stain. <b>B</b>: LC-MS/MS trace of the fusion protein GST-PfMCM2 containing the S13 to alanine mutation following phosphorylation with PfCK2 indicating the phosphorylation of residue Y16. Also shown is the fragmentation table (detected b-ions and y-ions are represented respectively in bold red and bold blue). <b>C</b>: N-terminal sequence of PfMCM2 protein showing the phospho-peptide identified in the LC-MS/MS analysis that contains the tyrosine phosphorylated residue (in red).</p

Autophosphorylation of PfCK2 regulates kinase activity.

<p>The activity of PfCK2α and a mutant PfCK2α where threonine 63 was mutated to alanine (T63A) was tested in <i>in vitro</i> kinase assays using α-casein as a substrate. <b>A</b>: Example of the <i>in vitro</i> kinase assay with PfCK2α and the T63A mutant. Top panel: autoradiograph, bottom panel: Coomassie stain. <b>B</b>: kinase activity quantification. Date represents the mean ± S.E.M (n = 3) <b>C</b>: LC-MS/MS trace of PfCK2 identifying T63 phosphorylation from a shizont stage lysate of <i>P. falciparum</i>. Indicated are the b-ions and b-ions (−98daltons) that were identified in the LC-MS/MS spectra. Also shown is the hypothetical fragmentation table where the ions that were identified in the LC-MS/MS spectra are shown in red.</p

Structural analysis of PfCK2α inhibition by quinalizarin.

<p><b>A</b>: <i>In vitro</i> inhibition assay showing the affect of various concentrations of quinalizarin on the activity of human protein kinase CK2α (red) and PfCK2α (blue). Date represents the mean ± S.E.M (n = 3) <b>B</b>: Superimposition of the calculate <i>in silico</i> homology model for PfCK2α (purple) with the <i>Zea mays</i> protein kinase CK2α crystal structure (green, PDB code: 3FL5, resolution 2.30 Å); <b>B</b>: Superimposition of the molecular docking of the PfCK2 homology model with quinalizarin (purple) and the co-crystal structure of <i>Z. mays</i> protein kinase CK2α and quinalizarin (green); non-conserved residues are indicated in bold.</p

Phylogenetic analysis of <i>Plasmodium</i> CDC20.

<p>A. Amino-acid sequence of a <i>Plasmodium berghei</i> putative CDC20. WD repeats are shown in bold and underlined. Other motifs (highlighted) are the KEN box, RVL cyclin binding motif and IR motif. B. WD domains from various eukaryotic Cdc20 and Cdh1 homologues were aligned and used to draw a phylogenetic tree. The <i>S. cerevisiae</i> meiotic APC/C regulator (Ama1) was used as an out group. Four clusters are apparent. Two correspond to previously described Cdc20 and Cdh1 protein families. A third includes homologues from <i>Trypanosomatidae</i> species. The fourth cluster includes all the identified CDC20/CDH1 homologues from <i>Plasmodium</i> species.</p

Summary of phenotypes in mutants of <i>cdpk4, map2</i> and <i>cdc20</i>.

<p><i>Cdpk4</i> mutants have been shown to arrest DNA synthesis after activation, whereas <i>cdc20</i> mutants show a similar phenotype to <i>map2</i> mutants.</p

CDC20-GFP protein expression in activated male gametocytes.

<p>High CDC20-GFP intensity was observed in activated male gametocytes and co-localised with Hoechst nuclear staining in both the microgametocyte body and exflagellating microgametes (arrows). Bar = 5 µm.</p