Label-free quantitative analysis of PfCK1 interacting proteins.

<p>Volcano plot representing the logarithmic ratio of protein LFQ intensities in the CK1/3D7 experiments plotted against negative logarithmic p-values of the <i>t</i> test performed from triplicates (FDR threshold = 0.1, S0 = 0.5). A hyperbolic curve separates specific CK1-interacting proteins (dark blue dots) from background (light blue dots). PfCK1 and PfRON3 are highlighted with a red square and a red triangle respectively. (B) Distribution of potential PfCK1 interactors across metabolic processes. Histogram representing the number of potential PfCK1 interactors distributed among the various metabolic processes described in the Metabolic Pathways of Malaria Parasites website (<a href="http://sites.huji.ac.il/malaria/" target="_blank">http://sites.huji.ac.il/malaria/</a>). The total number of proteins in the most represented pathway is represented in brackets. (C) Distribution of potential PfCK1 interactors across specific pathways in the transcription process. The histogram represents the number of potential PfCK1 interactors across the various pathways present in the “Transcription” process.</p

PfCK1 immunofluorescence assay on wild-type parasites and transgenic parasites expressing a GFP-tagged PfCK1.

<p>(A) 3D7 parasites were examined by immunofluorescence using an anti-PfCK1 serum labelled with rhodamine. DAPI was used to stain the nucleus and the scale bar represents 10 μm. A pre-immune serum used as a negative control did not yield any signal (not shown). (B) An erythrocyte culture infected with synchronised parasites expressing GFP-tagged PfCK1 from the endogenous locus was examined by immunofluorescence using an anti-GFP antibody. DAPI was used to stain the nucleus the scale bar represents 10 μm. As a negative control wild-type 3D7 parasites stained with the same anti-GFP antibody and did not yield any signal (not shown).</p

PfCK1 kinase activity in culture supernatants.

<p>(A) CK1 peptide phosphorylation by culture supernatants. (B) PfCK1 activity immunoprecipitated from culture supernatants of uninfected (uRBCs) or 3D7-infected (iRBC) red blood cells using an anti-PfCK1 antibody (α-PfCK1). A pre-immune serum (preim) was used as control. (C) Accumulation of casein kinase activity immunoprecipitated from supernatants of synchronised trophozoite cultures using antibodies against PfPK7, PfNek-1 and PfCK1. All three kinases immunoprecipitated from parasite extracts gave strong signals (not shown). (D) Secretion of endogenous GFP-tagged PfCK1. Anti-GFP Western blot showing GFP-tagged PfCK1 in the supernatant of transgenic parasites expressing GFP-tagged PfCK1, but not in supernatants of WT parasites. (E) Kinase activity immunoprecipitated with an anti-GFP antibody from culture supernatants. Lane 1: supernatant from a wild-type 3D7 culture; lane 2: supernatant from a transgenic line expressing GFP-tagged PfCK1.</p

Kinase activity of recombinant PfCK1 on potential interactors.

<p>(A) Autoradiograms and Coomassie blue stained gels of <i>in vitro</i> standard kinase reactions. Assays were performed with 500 ng of recombinant GST-PfCK1wild-type or with GST-PfCK1 K38M (“kinase dead mutant”). Both kinases were incubated with 3 μg of α-casein, or CK1 peptide substrate. GST-PfCK1 wt shows activity towards these 2 substrates whereas no signal was obtained with the control mutant. Lane 1: α-casein + CK1 substrate; lane 2: GST-PfCK1 WT + α-casein; Lane 3: GST-PfCK1 WT + CK1 substrate peptide; lane 4: GST-PfCK1[K38M]+ α-casein; lane 5: GST-CK1[K38M] + CK1 peptide substrate. (B) Phosphorylation of α-casein and PfNapL, but not PfNapS, by GST-PfCK1. Lane 1: GST-PfCK1 + α-casein; lane 2: GST-PfCK1 + His-PfNapL; lane 3: GST-PfCK1 + His-PfNapS. (C) Phosphorylation of PfRON3 and PfAlba4 by GST-PfCK1. Lane 1: GST-PfCK1+ α-casein; lane 2: GST-PfCK1 + GST; lane 3: GST-PfCK1 + His PfRON3; lane 4: GST-PfCK1 + GST-PfAlba 4.</p

Interaction between PfCK1 and PfNapL, PfCK2a or PfRON3.

<p><b>(A) Interaction between native PfCK1 and PfNapL, PfCK2a or PfRON3.</b> Immunoprecipitation was performed on protein extracts from transgenic parasites expressing GFP-tagged PfCK1 and from wild-type 3D7 parasites using GFP-trap beads. Detection of GFP, PfCK2a, PfNapL and PfRON3 was then performed by western-blot on the immunoprecipitates using the <i>ad hoc</i> antibodies. Lane 1: total extracts from PfCK1-GFP parasites; lane 2: total extracts from 3D7 WT parasites; lane 3: immunoprecipitates from PfCK1-GFP parasites; lane 4: immunoprecipitates from 3D7 WT parasites. <b>(B) Recombinant PfCK1 and PfCK2α interact <i>in vitro</i>.</b> GST-PfCK1 was incubated with His-PfCK2α and complexes containing the CK1 GST-tagged protein were then purified using glutathione agarose beads. The His-tagged proteins were detected by Western blot analysis using an anti-His antibody and the corresponding Coomassie blue-stained gels are shown. Lane1: Bound material after incubation of GST agarose beads with soluble His-PfCK2α; lane 2: bound material after incubation of GST-PfCK1 agarose beads with soluble His-PfCK2α; lane 3: soluble His-PfCK2α control. <b>(C) Recombinant PfCK1 and PfRON3 interact <i>in vitro</i>.</b> GST-PfCK1 was incubated with His-PfRON3 and complexes containing the CK1 GST-tagged protein were then purified using glutathione agarose beads. The His-tagged proteins were detected by Western blot analysis using an anti-His antibody and the corresponding Coomassie blue-stained gels are shown. Lane 1: bound material after incubation of GST agarose beads with soluble His-PfRON3; lane 2: bound material after incubation of GST-PfCK1 agarose beads with soluble His-PfRON3; lane 3: soluble His-PfRON3 control.</p

PfCK1 expression and kinase activity.

<p>(A) PfCK1 protein was detected by western blot using a peptide-derived anti-PfCK1 antibody. R: Rings; T: trophozoites; S: Schizonts; G: Gametocytes. Detection of 2-Cys-peroxiredoxin was used as a loading control (bottom panel). (B) Casein kinase activity immunoprecipitated with an anti-PfCK1 antibody from parasite extracts from a mixed asexual culture. Kinase assays were set up with casein as a substrate. Lane 1 (“-“), no immunoprecipitated added; lane 2 (“Preim”); immunoprecipitate obtained with a pre-immune serum lane 3 (“CK1”), immunoprecipitate obtained with the anti-PfCK1 antiserum. Reactions were subsequently analysed by SDS-PAGE and autoradiography. (C) Endogenously GFP tagged PfCK1. Western blot of parasite extracts from wild-type 3D7 parasites and parasites expressing GFP-tagged PfCK1 from the endogenous locus using anti-PfCK1, anti-GFP and anti-2-Cys-peroxiredoxin antibodies. (D) Kinase activity after immunoprecipitation from parasite extracts using an anti-GFP antibody. Kinase assays were set up with casein as a substrate. Lane 1, no immunoprecipitated added; lane 2, immunoprecipitate obtained from wild-type 3D7 parasites; lane 3, immunoprecipitate from parasites expressing GFP-tagged PfCK1 from the endogenous locus. Reactions were subsequently analysed by SDS-PAGE and autoradiography. (E) Endogenously His tagged PfCK1. Western Blot of parasite extracts from wild-type 3D7 parasites and parasites expressing His-tagged PfCK1 from the endogenous locus using anti-His and anti-PfCK1 antibodies. (F) Kinase activity after immunoprecipitation from parasite extracts using an anti-His antibody. Kinase assays were set up with casein as a substrate. Lane 1, immunoprecipitate obtained from wild-type 3D7 parasites; lane 2, immunoprecipitate from parasites expressing His-tagged PfCK1 from the endogenous locus. Reactions were subsequently analysed by SDS-PAGE and autoradiography.</p

Localisation of PfCK1 in micronemes.

<p>(A) Localisation of PfCK1 and PfRON3 in schizonts and segmenters. DAPI was used to stain the nucleus and the scale bar represents 10μm. (B) Localisation of PfCK1 and AMA-1 in merozoites. DAPI was used to stain the nucleus and the scale bar represents 1μm.</p

Characterization of <i>Plasmodium falciparum</i> Atypical Kinase PfPK7^– Dependent Phosphoproteome

PfPK7 is an “orphan” kinase displaying regions of homology to multiple protein kinase families. PfPK7 functions in regulating parasite proliferation/development as evident from the phenotype analysis of knockout parasites. Despite this regulatory role, the functions of PfPK7 in signaling pathways are not known. To better understand PfPK7-regulated phosphorylation events, we performed isobaric tag-based quantitative comparative phosphoproteomics of the schizont and segmenter stages from wild-type and <i>pfpk7</i>^<i>‑</i> parasite lines. This analysis identified 3,875 phosphorylation sites on 1,047 proteins. Among these phosphorylation events, 146 proteins with 239 phosphorylation sites displayed reduction in phosphorylation in the absence of PfPK7. Further analysis of the phosphopeptides revealed three motifs whose phosphorylation was down regulated in the <i>pfpk7</i>^<i>–</i> cell line in both schizonts and segmenters. Decreased phosphorylation following loss of PfPK7 indicates that these proteins may function as direct substrates of PfPK7. We demonstrated that PfPK7 is active toward three of these potential novel substrates; however, PfPK7 did not phosphorylate many of the other proteins, suggesting that decreased phosphorylation in these proteins is an indirect effect. Our phosphoproteomics analysis is the first study to identify direct substrates of PfPK7 and reveals potential downstream or compensatory signaling pathways

Characterization of <i>Plasmodium falciparum</i> Atypical Kinase PfPK7^– Dependent Phosphoproteome

PfPK7 is an “orphan” kinase displaying regions of homology to multiple protein kinase families. PfPK7 functions in regulating parasite proliferation/development as evident from the phenotype analysis of knockout parasites. Despite this regulatory role, the functions of PfPK7 in signaling pathways are not known. To better understand PfPK7-regulated phosphorylation events, we performed isobaric tag-based quantitative comparative phosphoproteomics of the schizont and segmenter stages from wild-type and <i>pfpk7</i>^<i>‑</i> parasite lines. This analysis identified 3,875 phosphorylation sites on 1,047 proteins. Among these phosphorylation events, 146 proteins with 239 phosphorylation sites displayed reduction in phosphorylation in the absence of PfPK7. Further analysis of the phosphopeptides revealed three motifs whose phosphorylation was down regulated in the <i>pfpk7</i>^<i>–</i> cell line in both schizonts and segmenters. Decreased phosphorylation following loss of PfPK7 indicates that these proteins may function as direct substrates of PfPK7. We demonstrated that PfPK7 is active toward three of these potential novel substrates; however, PfPK7 did not phosphorylate many of the other proteins, suggesting that decreased phosphorylation in these proteins is an indirect effect. Our phosphoproteomics analysis is the first study to identify direct substrates of PfPK7 and reveals potential downstream or compensatory signaling pathways

Identification of putative phosphosites and parasite kinase involved in modification of PfRh4 cytoplasmic tail.

<p>(A) <i>In vitro</i> kinase assays of wildtype and putative phosphosite mutations in PfRh4 cytoplasmic domains. The amino acid sequence of the PfRh4 cytoplasmic tail is shown with serines and tyrosines highlighted with the residue number. Each potential phosphosite on the PfRh4 tail was individually mutated to alanine. The all 4 lane has mutations in S1667A, S1674A, Y1680A and Y1684A and the all 5 lane has S1652A, S1667A, S1674A, Y1680A and Y1684A putative kinase sites mutated. The phosphorylation signal was quantitated and adjusted for protein loading. The loading-adjusted mutant phosphorylation signals were divided by the wildtype and plotted as a percentage of the wildtype signal (Y-axis). Autoradiograph of proteins after incubation in the <i>in vitro</i> phosphorylation assay and Coomassie gel from which protein loading was quantitated are shown in lower panels. Lane labels (X-axis) denote residues mutated to alanine. Mean percentage of wildtype phosphorylation +1 standard error of the mean are displayed. Data was averaged from four experiments performed on separate days. (B) Dosage-response curve for PfRh4 tail phosphorylation by merozoite lysate in the presence of increasing concentrations of the CK2 inhibitor TBB. PfRh4 tail phosphorylation was quantitated after incubation in <i>in vitro</i> phosphorylation assay with TBB. The phosphorylation signal for each condition was adjusted to reflect the average amount of protein loaded across each condition, determined by densitometry of the Coomassie brilliant blue stained gel. Y-axis represents loading-adjusted phosphorylation signal as a percentage of phosphorylation in the presence of DMSO (control). Autoradiograph of wildtype GST-fused PfRh4 proteins after incubation in the <i>in vitro</i> phosphorylation assay. X-axis indicates the TBB concentration with which the phosphorylation assay was incubated or DMSO. (C) <i>In vitro</i> kinase assays of PfRh and EBL cytoplasmic tails. The phosphorylation signal was quantitated and adjusted for protein loading. Autoradiograph of proteins after incubation in the <i>in vitro</i> phosphorylation assay and Coomassie brilliant blue stained gel from which protein loading was quantitated are shown. Data was averaged from four experiments performed on separate days (right panel) and standard error of the mean is shown. The following sites were mutated: EBA140 (S1159A, S1168A, T1173A), EBA175 (T1466A, mut A) and (S1489A, mut B) and in combination (mut A and B), EBA181 (S1528A, S1553A, S1557A, T1564A), PfRh2a (S3128A) and PfRh2b (S3233).</p