β-arrestins are not involved in ERK5 activation by Gq-coupled muscarinic receptors.

<p>(A) CHO cells stably expressing <i>wild-type</i> muscarinic M3 receptor (CHO-M3 cells) were transfected with cDNAs encoding for HA-ERK5 and either pcDNA3, Gαq, β-arrestin1-Flag or β-arrestin2-GFP. Twenty-four hours after transfection, cells were serum-starved for 2h and stimulated with carbachol (10µM) for the indicated times. ERK5 phosphorylation was assessed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084174#pone-0084174-g002" target="_blank">Figure 2</a>. Data (mean +/- SEM of 3 independent experiments) were normalised using HA-ERK5 as loading control and expressed as fold-induction over basal conditions (*p<0.05, **p<0.005, two-tailed T-test). Gαq, β-arrestin1-Flag and β-arrestin2-GFP, and α-tubulin expression levels were assessed with specific antibodies. (B) NIH-3T3-M1 cells were transfected with siRNA oligonucleotides targeting β-arrestin2 or non-targeting scrambled oligonucleotides (100nM) as detailed in the Materials and Methods section. After 72h of incubation, cells were serum-starved for 5h and challenged with carbachol (10µM) for the indicated times. Endogenous ERK5 phosphorylation was assessed in cell lysates with a phospho-ERK5 specific antibody. Total ERK5 appears as a double band corresponding to basal (lower) and hyper-phosphorylated (upper) kinase. A representative blot for 3 independent experiments with similar results is shown. (C) β-arrestin2 expression levels were also determined and quantified to estimate the overall efficiency of protein silencing. Data (mean +/- SEM of 3 independent experiments) were normalised using α-tubulin as loading control and expressed as the relative difference (%) to β-arrestin2 protein levels in scrambled siRNA-treated cells.</p

Gq-coupled muscarinic receptor-induced activation of the ERK5 pathway does not require receptor internalization.

<p>(A) CHO cells stably overexpressing <i>wild-type</i> muscarinic M3 receptor or internalization-deficient M3 receptor (SASS motif mutant, characterised in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084174#B10" target="_blank">10</a>]) were transfected with HA-ERK5. Twenty-four hours after transfection, cells were serum-starved for 2h and stimulated with carbachol (10µM). HA-ERK5 was immunoprecipitated with an anti-HA agarose-conjugated antibody as detailed in the Materials and Methods section. ERK5 phosphorylation was assessed in the immunoprecipitate using a phosphospecific antibody. Data (mean +/- SEM of 3 independent experiments) were normalised using HA-ERK5 as loading control and expressed as fold-induction over basal conditions (*p<0.05, two-tailed T-test). (B) Quantification of cell surface receptor density was performed through [³H]-NMS binding at 4°C. The two cell types utilised in (A) were serum-starved for 2h and stimulated with carbachol (100µM) for 30 minutes. Data were normalised to unspecific binding (atropine treatment) and binding percentage was expressed as the mean +/- SEM of 3 independent experiments.</p

Gq-coupled muscarinic receptor-induced activation of the ERK5 pathway does not require receptor phosphorylation.

<p>Different stable lines of CHO cells (previously characterised in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084174#B11" target="_blank">11</a>]) that stably overexpress <i>wild-type</i> or phosphorylation-deficient muscarinic M3 receptor (A) and <i>wild-type</i> or phosphorylation-deficient muscarinic M1 receptor (B) were utilised. All cell lines were transfected with HA-ERK5. Twenty-four hours after transfection, cells were serum-starved for 2h and stimulated with acetylcholine (100µM) for the indicated times (A) or incubated for 15min with acetylcholine at various concentrations (B). ERK5 phosphorylation was assessed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084174#pone-0084174-g002" target="_blank">Figure 2</a>. Data (mean +/- SD of 2-3 independent experiments) were normalised using HA-ERK5 as loading control and expressed as fold-induction over basal conditions or over maximum activation (*p<0.05, **p<0.005; two-tailed T-test). </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

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

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

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

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

Phosphorylation of plasmodial SR proteins by immunoprecipitated PfCLKs.

<p>A. Kinase activity assays were deployed to detect phosphorylation of recombinant PfSRSF12 and PfSFRS4 (∼73 and 65 kDa, respectively; indicated by arrows) by two or more of the PfCLKs (autoradiogram; upper panel). B. The N-terminal part (∼95 kDa; indicated by arrows), but not the C-terminal part (86 kDa) of recombinant PfSF-1 was phosphorylated by immunoprecipitated PfCLKs. An additional phosphorylation signal of truncated N-terminal PfSF-1 was visible at approximately 60 kDa. C. MaBP-tag alone (43 kDa) as substrate was used as negative control. Shown here is an assay using PfCLK-3-specific immunoprecipitate, similar results were obtained with immunoprecipitates of other PfCLKs (not shown). Coomassie blue staining (lower panels) of radiolabelled SDS gels was used as a loading control.</p

Subcellular localization of PfCLK-3 and PfCLK-4 in the blood and gametocyte stages.

<p>Mixed asexual blood stage cultures containing trophozoites (TZ) and schizonts (SZ) and mature gametocyte (GC) cultures were fixed with methanol and prepared for IFA, using rat antisera against PfCLK-3 and mouse antisera against PfCLK-4 (green). The parasite nuclei were highlighted by Hoechst staining (blue). The asexual blood stages were labelled with rabbit antisera against PfMSP-1 and gametocytes with rabbit antisera against Pfs230 (red). Bar, 5 µm.</p