rabbit anti phospho pka substrate motif  (Cell Signaling Technology Inc)

Journal: PLoS Biology

Article Title: Phosphodiesterase beta is the master regulator of cAMP signalling during malaria parasite invasion

doi: 10.1371/journal.pbio.3000154

Figure Lengend Snippet: (A) Western blot analysis of DMSO- and RAP-treated mature cycle 0 PfPDEβ ΔcatHA schizonts, probed with antibodies specific for the phosphorylated generic consensus PKA substrate motif. The blot was reprobed with an anti-HA antibody to monitor disruption of PfPDEβ-HA expression, as well as an antibody to MyoA as a loading control. (B) Changes in phosphorylation and protein abundance between RAP-treated (PfPDEβ-null, KO) and mock-treated (WT) Compound 2–arrested PfPDEβ ΔcatHA schizonts. Left panel: peptide intensity (log 10 ) plotted against log 2 fold change for 5,374 phosphosites, with significantly altered sites (Welch-corrected t test) in dark grey. Seven phosphosites from the PDEβ N-terminal domain (red) and four significantly up-regulated phosphosites in ACβ and CDPK1 (green), as well as MyoA and AMA1 (blue), are highlighted. Right panel: changes in protein abundance, with PfPDEβ, hDHFR, mTOR, and FKBP1A highlighted. (C) Sequence logo showing consensus sequence surrounding phosphosites (position 0) significantly increased in the PfPDEβ-null samples. (D) Motif analysis showing the six motifs most enriched in the PfPDEβ-null samples by 1D annotation analysis (green) and two control kinase motifs (red). CDPK1 and CRK4 motifs used are described in Materials and methods. Data show mean log 2 fold changes. Error bars, SEM. Numbers in parentheses denote the frequency of the occurrence of the motif in phosphosites significantly up-regulated in the PfPDEβ-null/total number of phosphosites with that motif. (E) Presentation of GO terms dysregulated in PfPDEβ-null schizonts. Bars show numbers of phosphosites up- (green) and down-regulated (red) in the PfPDEβ-null schizonts compared with wild type. Light shades denote sites significantly different by Welch t test, and bright colours denote sites >2-fold changed. Hatched bars mark phosphosites with a minimal PKA consensus motif (K/R, x, S/T). The numbers of proteins in each group are indicated on the right. ACβ, adenylyl cyclase β; AMA1, apical membrane antigen-1; CDPK1, calcium-dependent protein kinase 1; CRK4, cdc2-related protein kinase 4; FKBP1A, FK506-binding protein 1A; GO, gene ontology; HA, haemagglutinin; hDHFR, human dihydrofolate reductase; KO, knockout; K/R, lysine/arginine; mTOR, mechanistic target of rapamycin; MyoA, myosin A; PDEβ, phosphodiesterase β; PfPDEβ, Plasmodium falciparum phosphodiesterase β; PKA, cAMP-dependent protein kinase; RAP, rapamycin; S/T, serine/threonine; WT, wild type; x, any amino acid; 1D, one-dimensional.

Article Snippet: A rabbit monoclonal antibody specifically reacting with phosphorylated PKA substrate consensus motif (R,K/R,X,pS/pT) was purchased from Cell Signaling Technology.

Techniques: Western Blot, Disruption, Expressing, Control, Phospho-proteomics, Quantitative Proteomics, Sequencing, Membrane, Binding Assay, Knock-Out

Journal: PLoS Biology

Article Title: Phosphodiesterase beta is the master regulator of cAMP signalling during malaria parasite invasion

doi: 10.1371/journal.pbio.3000154

Figure Lengend Snippet: (A) The PDE inhibitor BIPPO phenocopies the PfPDEβ-null post-invasion phenotype. Giemsa-stained blood films showing the morphology of PfPDEβ ΔcatHA parasites at different time points in cycle 1, following either mock or RAP treatment of the parasites in cycle 0, compared to mock-treated parasites exposed to the PDE inhibitor BIPPO immediately following invasion. (B) The PKA inhibitor H89, but not the PKG inhibitor Compound 2, rescues BIPPO-treated early ring stages. The plots were generated by microscopic analysis of Giemsa-stained smears from ring stage cultures (18–22 hpi) treated with the indicated compounds. Kinase inhibitors (H89 and C2) were added at 1–5 hpi and BIPPO at 2–6 hpi. Scale bar, 5 μm. Data presented are mean counts (evaluated by three independent researchers) from two independent experiments, with >100 parasites counted per condition. Error bars, SEM. *, significant by unpaired t test ( p -value = 0.0005); n.s., not significant ( p -value = 0.8508). (C) The PDE inhibitor BIPPO induces PKA-dependent phosphorylation in ring stages. Western blot of total protein from ring stages (12–16 hpi) treated for 90 minutes with various inhibitors: BIPPO (2 μM), H89 (30 μM), C2 (2 μM). Lane 1 = no inhibitor control, lane 2 = BIPPO only, lane 3 = BIPPO + H89, lane 4 = BIPPO + C2. The blot was probed with an antibody to phosphorylated PKA substrate motif. The gel was stained for total protein prior to blotting to show equal loading (right panel). BIPPO, 5-Benzyl-3-isopropyl-1 H -pyrazolo[4,3- d ]pyrimidin-7(6 H )-one; hpi, hours post invasion; PDE, phosphodiesterase; PfPDEβ, Plasmodium falciparum phosphodiesterase β; PKA, cAMP-dependent protein kinase; pS/pT, phosphoserine or phosphothreonine; RAP, rapamycin; R/K, arginine or lysine.

Article Snippet: A rabbit monoclonal antibody specifically reacting with phosphorylated PKA substrate consensus motif (R,K/R,X,pS/pT) was purchased from Cell Signaling Technology.

Techniques: Staining, Generated, Phospho-proteomics, Western Blot, Control