Journal:

Article Title: ?-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins

doi: 10.1073/pnas.211572798

Figure Lengend Snippet: Enhanced binding of βarr1 to phosphorylated Dvl. (A) HEK-293 cells were transfected with Myc-Dvl2 and FLAG-βarr1, and cell extracts were immunoprecipitated with anti-Myc antibody directed against Dvl2. Immunoprecipitated Dvl2 was phosphorylated in the presence of [γ-32P]ATP by endogenous Dvl2-associated kinase(s). Immunoprecipitated Dvl2 was processed for autoradiography (Upper) or incubated with His-βarr1 for >3 h at 4°C. Washed Dvl2 immunoprecipitates were analyzed by immunoblot with anti-His antibody to detect associated βarr1 (Lower). (B) MBP-Dvl1 was phosphorylated by protein kinase CKII in the presence of ATP. Phosphorylated and unphosphorylated control MBP-Dvl1 was incubated with His-βarr1 for >3 h at 4°C, and associated βarr1 was detected by immunoblot with anti-His antibody directed against βarr1. (C) MBP-Dvl1 was phosphorylated to various stoichiometric levels by protein kinase CKII. Phosphorylated MBP-Dvl1 was then either eluted in sample buffer and processed for autoradiography (Inset Upper) or incubated with His-βarr1. MBP-Dvl1-associated βarr1 was detected by immunoblot with anti-His antibody directed against βarr1 (Inset Lower). The relative amounts of βarr1 bound to MBP-Dvl1 phosphorylated by protein kinase CKII were quantified as fold increases over that bound to unphosphorylated MBP-Dvl1. Results are the mean ± SEM of three independent experiments.

Article Snippet: MBP-Dvl1 was washed with protein kinase CKII buffer (20 mM Tris, pH 7.5/50 mM KCl/2 mM MgCl 2 ) and phosphorylated with protein kinase CKII (New England Biolabs) and [γ- 32 P]ATP (60 μM; final concentration, 400 cpm/pmol) at 30°C for 30 min. Phosphorylated MBP-Dvl1 was visualized by exposure of dried gels to film.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Autoradiography, Incubation, Western Blot

Journal: mBio

Article Title: CK2 Phosphorylation of Human Papillomavirus 16 E2 on Serine 23 Promotes Interaction with TopBP1 and Is Critical for E2 Interaction with Mitotic Chromatin and the Viral Life Cycle

doi: 10.1128/mBio.01163-21

Figure Lengend Snippet: (A) GST-TopBP1 or GST-NEDD4 (0.65 pmol) was incubated with 11 pmol E2 and incubated at 4°C for 1 h with rotation. GST pulldowns followed by Western blotting for TopBP1 (top blot) and E2 (bottom blot) were then carried out. (B) The GST pulldown was repeated as in panel A following 1 h at 30°C with CK2 and controls. (C) Lambda phosphatase was added to the CK2 reaction mixture, and GST pulldown assays were carried out as in panel A. E2-specific antibody TVG261 (ab17185) was used for Western blotting in all three experiments (A to C). Figure S1A to S1C in the supplemental material summarizes quantitation of repeat experiments. In the input lanes, only E2 was added. The TopBP1 pulldown demonstrates equivalent levels of TopBP1 in each reaction mixture containing TopBP1.

Article Snippet: After 1 h, the beads were incubated with 1 μl CK2 enzyme and 1× CK2 reaction buffer (catalog no. P6010S; NEB Inc.) supplemented with 200 μM ATP and 30 mM MgCl 2 and rotated for 1 h at 30°C.

Techniques: Incubation, Western Blot, Quantitation Assay

Journal: mBio

Article Title: CK2 Phosphorylation of Human Papillomavirus 16 E2 on Serine 23 Promotes Interaction with TopBP1 and Is Critical for E2 Interaction with Mitotic Chromatin and the Viral Life Cycle

doi: 10.1128/mBio.01163-21

Figure Lengend Snippet: (A) siRNA knockdown of CK2α and/or CK2α′. Scr control siRNA was used in lanes 1 and 5. The top panels demonstrate the input proteins that were used in the immunoprecipitation (IP) with pS23-Ab (an antibody raised against an E2 peptide containing a phosphorylated serine 23). Please note the CK2α blot is independent of the other inputs but is run with the same protein extracts. The IP was blotted for E2 which is clearly detected in the Scr control (lane 5), but not in the CK2 knockdown cells (lanes 2 to 4). (B) The indicated cells were treated with DMSO (lanes 1 and 2) or the CK2 inhibitor CX4945 (lanes 3 and 4), and Western blotting details the levels of TopBP1 and E2 in the treated cells (top blots). Immunoprecipitation (IP) with TopBP1 demonstrates a pulldown of TopBP1 and E2 (lane 2) that is abrogated by CX4945 (lane 4) (middle blots). IP with pS23-Ab pulled down E2 in control cells (lane 2) that was abolished by CX4945 (lane 4). (C) CK2a siRNA knockdown (lane 4) disrupted the E2-TopBP1 interaction as there was a reduced co-IP of E2 in the absence of CK2α (lane 7). (D) CK2α′ knockdown (lane 3) disrupted the E2-TopBP1 interaction as there was a reduced co-IP of E2 in the absence of CK2α′ (lane 5). Please note that lanes 1 to 3 are from the same gel and the same exposure with a lane removed. (E) Staining of U2OS E2-WT (top panels) and U2OS E2-S23A (bottom panels) with pS23-Ab. Left-hand panels are antibody only, right-hand panels are antibody plus DAPI. There was no signal generated with secondary antibody only, and no signal detected in U2OS-Vec control when the primary antibody was included. HPV16 E2 B9 monoclonal antibody was used for Western blotting in panels A and B. E2-specific antibody TVG261 (ab17185) was used for Western blotting in panels C and D. Figure S2 A and B summarizes quantitation for repeat experiments of panels C and D, respectively. An asterisk indicates an antibody band.

Article Snippet: After 1 h, the beads were incubated with 1 μl CK2 enzyme and 1× CK2 reaction buffer (catalog no. P6010S; NEB Inc.) supplemented with 200 μM ATP and 30 mM MgCl 2 and rotated for 1 h at 30°C.

Techniques: Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Staining, Generated, Quantitation Assay

Journal: mBio

Article Title: CK2 Phosphorylation of Human Papillomavirus 16 E2 on Serine 23 Promotes Interaction with TopBP1 and Is Critical for E2 Interaction with Mitotic Chromatin and the Viral Life Cycle

doi: 10.1128/mBio.01163-21

Figure Lengend Snippet: (A) Top blots with lanes 1 to 3 are Western blots of extracts from the indicated stable N/Tert-1 cell lines. Bottom blots with lanes 4 to 6 are Western blots of a TopBP1 immunoprecipitation (IP) of the indicated extracts. TopBP1 co-IPs E2-WT but not E2-S23A. (B) The extracts in the top blots (Input) were immunoprecipitated with pS23-Ab, and E2 is pulled down by this antibody (bottom blot, lane 2). The CK2 inhibitor CX4945 abrogates this pulldown (lane 5). (C) The extracts from panel B were immunoprecipitated with TopBP1, and both E2-WT and E2-S23D co-IP with TopBP1 (lanes 2 and 3). Treatment with the CX4945 abrogates the interaction between TopBP1 and E2-WT (lane 6), but not E2-S23D (lane 5). (D) Organotypic raft cultures of N/Tert-1 (top panels) and N/Tert-1+HPV16 (bottom panels) were stained with pS23-Ab. There is no specific staining in N/Tert-1 cells, but there is clear staining in N/Tert-1+HPV16. (Left panels) pS23-Ab only, (right panels) pS23-Ab plus DAPI staining. An asterisk indicates an antibody band. (E) Organotypic raft cultures of N/Tert-1 (top panels) and N/Tert-1+HPV16 (bottom panels) stained with CK2 antibody. HPV16 E2 B9 monoclonal antibody was used for Western blotting in panels A to C.

Article Snippet: After 1 h, the beads were incubated with 1 μl CK2 enzyme and 1× CK2 reaction buffer (catalog no. P6010S; NEB Inc.) supplemented with 200 μM ATP and 30 mM MgCl 2 and rotated for 1 h at 30°C.

Techniques: Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Staining

Journal: eNeuro

Article Title: Mammalian FMRP S499 Is Phosphorylated by CK2 and Promotes Secondary Phosphorylation of FMRP

doi: 10.1523/ENEURO.0092-16.2016

Figure Lengend Snippet: Agents used in N2a-based pharmacological screens.

Article Snippet: rFMRP was diluted to 11.7 µ m in CK2 kinase buffer (NEBuffer for Protein Kinases, #B6022; New England Biolabs, Ipswich, MA) and incubated with 4 m m ATP with or without active CK2 (New England Biolabs, #P6010).

Techniques:

Journal: eNeuro

Article Title: Mammalian FMRP S499 Is Phosphorylated by CK2 and Promotes Secondary Phosphorylation of FMRP

doi: 10.1523/ENEURO.0092-16.2016

Figure Lengend Snippet: CK2 phosphorylates mammalian FMRP S499 in vitro. rFMRP S500 or S500D was incubated with or without recombinant CK2 for 30 min. Samples were resolved by SDS-PAGE and probed with pFMRP S499, tFMRP, or CK2a1 antibodies. Only rFMRP S499 incubated with CK2 showed a positive phosphosignal.

Article Snippet: rFMRP was diluted to 11.7 µ m in CK2 kinase buffer (NEBuffer for Protein Kinases, #B6022; New England Biolabs, Ipswich, MA) and incubated with 4 m m ATP with or without active CK2 (New England Biolabs, #P6010).

Techniques: In Vitro, Incubation, Recombinant, SDS Page

Journal: eNeuro

Article Title: Mammalian FMRP S499 Is Phosphorylated by CK2 and Promotes Secondary Phosphorylation of FMRP

doi: 10.1523/ENEURO.0092-16.2016

Figure Lengend Snippet: CK2 phosphorylates mammalian FMRP S499. A , Immunoblots for 3-h treatment of N2a cells with vehicle (DMSO), D4476 (25 µ m ), IC261 (20 µ m ), PHA-767491 (5 µ m ), DRB (50 µ m ), TBB (25 µ m ), or βARK (200 µ m ) followed by Western blot for protein indicated on the left. B , Quantification of pFMRP/tFMRP signal in A . ns, not significant (Kruskal–Wallis one-way ANOVA analysis [ H (8) = 4.56, p = 0.8034], n = 4). C , Immunoblot for 24-h treatment of N2a cells with the same agents listed in A . D , Quantification of pFMRP/tFMRP signal in C (Kruskal–Wallis one-way ANOVA [ H (8) = 7.239, p = 0.5111], n = 4, error bars = SEM). E , Baseline, untreated N2a cells were collected at time 0, and the remainder of the cells were treated with either DMSO or CX-4945 (5 or 1 µ m ) for 24 h. tFMRP and pFMRP S499 signals increased in DMSO-treated samples; however, only tFMRP increased in CX-treated samples, thereby causing a significant reduction in relative FMRP S499 phosphorylation. All immunoblot signals are from the same membrane; however, intervening lanes have been removed for clarity. F , Quantification of pFMRP S499 to tFMRP ratio from D ; one-way ANOVA, n = 4, error bars = SEM, * p < 0.05. G , HEK293 cells were collected at baseline (time 0) or treated for 24 h with DMSO or CX-4945. H , CX-4945 significantly reduced FMRP S499 phosphorylation compared with DMSO (one-tailed Mann–Whitney test, p = 0.0143). I , J , Mouse cortical neurons at 7 d in vitro treated with 1 µ m CX-4945 for 24 h exhibited a significant reduction in FMRP S499 phosphorylation compared with DMSO-treated neurons (one-tailed Mann–Whitney test, p = 0.05).

Article Snippet: rFMRP was diluted to 11.7 µ m in CK2 kinase buffer (NEBuffer for Protein Kinases, #B6022; New England Biolabs, Ipswich, MA) and incubated with 4 m m ATP with or without active CK2 (New England Biolabs, #P6010).

Techniques: Western Blot, One-tailed Test, MANN-WHITNEY, In Vitro

Journal: eNeuro

Article Title: Mammalian FMRP S499 Is Phosphorylated by CK2 and Promotes Secondary Phosphorylation of FMRP

doi: 10.1523/ENEURO.0092-16.2016

Figure Lengend Snippet: FMRP S499 rephosphorylation kinetics after CX-4945 treatment and washout mirrors a known CK2 target. A , N2a cells were collected either at baseline or 24 h after DMSO or CX-4945 treatment. Additional samples were collected after 24-h treatment and washout of DMSO or CX-4945 for different time periods. B , Quantification of [p/t]FMRP or [p/t]AKT compared with baseline. Changes in ratios were quantified by two-way ANOVA followed by Dunnett’s multiple comparisons post hoc test, n = 4 per data point, error bars = SEM. **** p < 0.0001; *** p < 0.001; * p < 0.05.

Article Snippet: rFMRP was diluted to 11.7 µ m in CK2 kinase buffer (NEBuffer for Protein Kinases, #B6022; New England Biolabs, Ipswich, MA) and incubated with 4 m m ATP with or without active CK2 (New England Biolabs, #P6010).

Techniques:

Journal: eNeuro

Article Title: Mammalian FMRP S499 Is Phosphorylated by CK2 and Promotes Secondary Phosphorylation of FMRP

doi: 10.1523/ENEURO.0092-16.2016

Figure Lengend Snippet: Alternative model for FMRP phosphorylation and regulation of translation. FMRP is first phosphorylated by CK2 on S499. FMRP S499 phosphorylation is permissive for secondary phosphorylation of FMRP on serine/threonine residues, presumably downstream of mGluR-I, by unknown kinases. Secondary phosphorylation of FMRP is counteracted by PP2A-mediated dephosphorylation. The dotted green and red lines are provisional and indicate that the relationship between FMRP’s phosphorylation status and protein translation is unknown.

Article Snippet: rFMRP was diluted to 11.7 µ m in CK2 kinase buffer (NEBuffer for Protein Kinases, #B6022; New England Biolabs, Ipswich, MA) and incubated with 4 m m ATP with or without active CK2 (New England Biolabs, #P6010).

Techniques: De-Phosphorylation Assay

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Measurement of the Multiple Activities of 26S Proteasomes

doi: 10.1007/978-1-4939-8706-1_19

Figure Lengend Snippet:

Article Snippet: Pool Sic1 fractions and dilute against 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10% glycerol, 1 mM DTT) Set up phosphorylation reaction (see Note 8 ) 10x Protein Kinase Buffer (NEB) 9 μL 500,000 U/mL CK2 (NEB) 7.5 μL Sic1 58.5 μL ATP, [γ-32P]- 6000Ci/mmol 10mCi/ml, 250 μCi (Perkin Elmer) 15 μL Open in a separate window Incubate at 30°C for 1 hour Remove unincorporated nucleotides by adding the phosphorylation reaction to a Bio-Spin 30 (Bio-Rad) column.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Protein Kinase CK2 Controls Ca V 2.1-Dependent Calcium Currents and Insulin Release in Pancreatic β-cells

doi: 10.3390/ijms21134668

Figure Lengend Snippet: CK2 phosphorylates Ca V 2.1. ( A ) An in vitro phosphorylation assay was carried out by incubating lysates (80 µg) of INS-1 cells with CK2 in the presence of DMSO (control) or 10 µM CX-4945. Proteins were subjected to SDS polyacrylamide gel electrophoresis and blotted onto a PVDF membrane. A representative autoradiography ( 32 P) of the blotted proteins and the corresponding immunoblot (IB) of the same membrane with an anti-Ca V 2.1 and an anti-tubulin-specific antibody is shown. ( B ) Comparison of the amino acid sequences of the Ca V 2.1 C-terminus from different species (GenBank protein IDs: zebra fish NP_001315637.1, mouse AAW56205.1, rat XP_017456671.1, dog XP_013977279.1, human AAB64179.1). Dashes indicate a missing amino acid and spacing has been adjusted to maximize the homology of the sequences. The highlighted amino acids indicate putative CK2 phosphorylation sites with the sequence S/TxxD/E ( C ) Peptides with the putative CK2 phosphorylation sites shown in B (based on the murine sequence) were spotted on a cellulose membrane. Filters were incubated in the absence (-) or the presence of CK2 (+) and [ 32 P]γATP. A representative autoradiography is shown. ( D ) An in vitro phosphorylation assay was carried out by incubating equal amounts of GST, GST–Ca V 2.1 (2177-2369) wild-type or double mutant S2362A/S2364A with CK2 and [ 32 P]γATP. A sample with CK2 alone (-) or with nucleolin, as an established CK2 substrate, served as controls. Samples were analyzed on a 12.5% SDS polyacrylamide gel and subjected to staining with Coomassie Brilliant Blue (CBB) followed by autoradiography ( 32 P). A representative autoradiography and the corresponding CBB staining is shown.

Article Snippet: After incubation, the filter was washed with kinase buffer and the phosphorylation reaction was started by the addition of CK2 holoenzyme and [ 32 P]γATP (10 μCi, Hartmann Analytic, Braunschweig, Germany) in 1 mL kinase buffer.

Techniques: In Vitro, Phospho-proteomics, Control, Polyacrylamide Gel Electrophoresis, Membrane, Autoradiography, Western Blot, Comparison, Sequencing, Incubation, Mutagenesis, Staining

Journal: International Journal of Molecular Sciences

Article Title: Protein Kinase CK2 Controls Ca V 2.1-Dependent Calcium Currents and Insulin Release in Pancreatic β-cells

doi: 10.3390/ijms21134668

Figure Lengend Snippet: CK2 binds to Ca V 2.1. ( A ) GST pull-down analysis of Ca V 2.1 and CK2. Equal amounts of GST–Ca V 2.1 (2177-2369) wild-type and double-mutant S 2362 A/S 2364 A coupled to GSH sepharose were incubated with in vitro-translated and [ 35 S]-methionine-labeled CK2α and CK2β proteins. Proteins bound to the affinity matrix were subjected to SDS polyacrylamide gel electrophoresis, stained with Coomassie Brilliant Blue (CBB), followed by autoradiography ( 35 S). A representative autoradiography and a corresponding CBB staining are shown. ( B ) Co-immunoprecipitation of Ca V 2.1 and CK2 from INS-1 cells. Five milligrams of extract from cells grown with 0 mM (-) or 10 mM (+) glucose were pre-cleared twice from non-specific binding proteins with protein G sepharose beads (ctrl). After the centrifugation of the beads, the supernatants were incubated with a rabbit Ca V 2.1 antibody coupled to protein G sepharose overnight. Bound proteins were extracted with sample buffer, separated in a 10% SDS polyacrylamide gel and blotted to a PVDF membrane. Ca V 2.1 and CK2α subunit were detected with a rabbit monoclonal antibody directed against Ca V 2.1 or the mouse monoclonal antibody 1A5 against CK2α. Ctrl: proteins bound to protein G sepharose; input: 2% of whole protein extract used for immunoprecipitation; IPP: proteins immunoprecipitated with Ca V 2.1-specific antibody.

Article Snippet: After incubation, the filter was washed with kinase buffer and the phosphorylation reaction was started by the addition of CK2 holoenzyme and [ 32 P]γATP (10 μCi, Hartmann Analytic, Braunschweig, Germany) in 1 mL kinase buffer.

Techniques: Mutagenesis, Incubation, In Vitro, Labeling, Polyacrylamide Gel Electrophoresis, Staining, Autoradiography, Immunoprecipitation, Binding Assay, Centrifugation, Membrane

Journal: International Journal of Molecular Sciences

Article Title: Protein Kinase CK2 Controls Ca V 2.1-Dependent Calcium Currents and Insulin Release in Pancreatic β-cells

doi: 10.3390/ijms21134668

Figure Lengend Snippet: Inhibition of CK2 increases insulin secretion and cytosolic Ca 2+ concentration in INS-1 cells. Prior to the experiments, cells were starved for 2 h in glucose-free medium. ( A ) Glucose-induced insulin secretion in the absence or presence of the CK2 inhibitors CX-4945 (10 µM) or quinalizarin (50 µM). Insulin levels in the supernatant were measured after 30 min. Data are expressed as means of three independent experiments with two technical replicates each. Statistical analysis was performed by using Student’s t -tests. * p < 0.05, ** p < 0.01. ( B ) Ca 2+ imaging experiments in the absence or in the presence of the CK2 inhibitor CX-4945 (10 µM). Measurements were started in Ca 2+ -free medium. Changes in the cytosolic Ca 2+ concentration were determined by Fura 2-AM (5 µM) imaging measurements and plotted versus time. Each trace represents the mean ± SEM of the fluorescence ratios (F 340/ F 380 ) obtained in three independent experiments of the total number of cells indicated in brackets. ( C ) Mean area under the curve (AUC) of the three independent experiments shown in B. AUC of the DMSO-treated cells was set as 100%. Statistical analysis was performed by using Student’s t -tests. * p < 0.05.

Article Snippet: After incubation, the filter was washed with kinase buffer and the phosphorylation reaction was started by the addition of CK2 holoenzyme and [ 32 P]γATP (10 μCi, Hartmann Analytic, Braunschweig, Germany) in 1 mL kinase buffer.

Techniques: Inhibition, Concentration Assay, Imaging, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Protein Kinase CK2 Controls Ca V 2.1-Dependent Calcium Currents and Insulin Release in Pancreatic β-cells

doi: 10.3390/ijms21134668

Figure Lengend Snippet: Knockdown of Ca V 2.1 expression abolishes the CX-4945-induced increase in the cytosolic Ca 2+ concentration and enhanced insulin secretion in INS-1 cells. ( A – C ): INS-1 cells were transfected with Ca V 2.1 siRNA or scrambled control siRNA (200 nM), together with the fluorescent transfection indicator siGlo for 48 h. ( A ) Ca 2+ imaging experiments after Ca v 2.1 knockdown. Prior to the measurement, the cells were starved for 2 h in glucose-free medium. Cells were then incubated with the CK2 inhibitor CX-4945 (10 µM) or the solvent DMSO as a control and insulin secretion was induced with glucose (10 mM). Calcium imaging was only done in those cells where the successful transfection was indicated by siGlo. Changes in [Ca 2+ ] were determined by Fura 2-AM (5 µM) measurements and plotted versus time. Each trace represents the mean ± SEM of fluorescence ratios ( F 340/380 ) of two or three independent experiments of the total number of cells indicated in brackets. ( B ) Cell culture supernatants of cells treated as described for A were collected and analyzed for secreted insulin by an ELISA assay. Determination was done in three independent experiments with two technical replicates each and values were normalized to the respective DMSO control. Data are expressed as means ± SEM. Statistical analysis was performed by using Student’s t -tests. * p < 0.05, ** p < 0.01. ( C ) Equal amounts of extracts from all cells were analyzed by SDS polyacrylamide gel electrophoresis and subsequent immunoblot analysis with anti-Ca V 2.1- or anti-hsp70-specific antibodies (left panels). A representative immunoblot is shown. Ratios between the arbitrary amount of Ca V 2.1 and the loading control hsp70 were quantified by densitometry (Ca V 2.1/hsp70) and compared with the corresponding control value normalized to 100% (right panel). Data are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by using Student’s t -tests. *** p < 0.001.

Article Snippet: After incubation, the filter was washed with kinase buffer and the phosphorylation reaction was started by the addition of CK2 holoenzyme and [ 32 P]γATP (10 μCi, Hartmann Analytic, Braunschweig, Germany) in 1 mL kinase buffer.

Techniques: Knockdown, Expressing, Concentration Assay, Transfection, Control, Imaging, Incubation, Solvent, Fluorescence, Cell Culture, Enzyme-linked Immunosorbent Assay, Polyacrylamide Gel Electrophoresis, Western Blot