Journal: Nature communications

Article Title: Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7

doi: 10.1038/ncomms6827

Figure Lengend Snippet: a) Schematic of the domain organization and location of LRRK2 constitutive phosphorylation sites. Domains include ankyrin (Ank), leucine-rich-repeat (LRR), Ras of complex proteins (ROC), C-terminal of ROC (COR), kinase and WD40 domains. Constitutive phosphorylation sites are clustered upstream of the LRR domain and crucial for binding to 14-3-3 proteins. Pathogenic mutations, shown in red (R1441C, Y1699C and G2019S; N1437S not shown), S910A/S935A, T1348N and K1906M, shown in black, are designed mutants used to block 14-3-3 binding, GTP/GDP binding and kinase activity respectively. b) RNAi screen against kinases to identify kinase regulators of LRRK2 at S935. The screen was performed in duplicate per siRNA pool and each value of ratio pS935/LRRK2 was converted with a Z-transformation, adjusted for date of assay. Hits were identified if both replicates were 3 standard deviation Z away from mean. CSNK1A1 and WEE1 were two candidates with adjusted Z < −3.0 in both duplicates (bottom left grey box). c) Western blot example from the RNAi screen identifying CSNK1A1 as the candidate kinase for S935 LRRK2. Recombinant LRRK2, purified from cells pre-treated with DMSO or LRRK2-IN1, were included in each blot as loading control to allow for normalization across blots. d) CSNK1A1 validated using single siRNAs and pooled siRNAs. Three of four single CSNK1A1 siRNAs showed that when CK1α was knocked down, S935 phosphorylation was also reduced. Representative blots from 3 independent experiments. NTC – non-targeting control, single CSNK1A1 siRNAs - #1, 2, 3, 4 (used at 6.25nM final concentration). e) Quantitation of blots in 1d. Graph shows mean +/− SEM (n=3) for relative CK1α and phosphorylated LRRK2 signals.

Article Snippet: Equal amounts of immunopurified LRRK2 per experiment were incubated with 100 μM non-radioactive ATP, 100 ng recombinant active CK1 isoforms (Signal Chem Cat# C64-10G, C65-10G, C66-10G, C68-11G, C68-10BG, C68-10CG) or WEE1 (Active Motif Cat# 31201) and with or without DMSO, LRRK2-IN1 and IC261, in a final volume of 20 μl in cold kinase buffer.

Techniques: Phospho-proteomics, Binding Assay, Blocking Assay, Activity Assay, Transformation Assay, Standard Deviation, Western Blot, Recombinant, Purification, Control, Concentration Assay, Quantitation Assay

Journal: Nature communications

Article Title: Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7

doi: 10.1038/ncomms6827

Figure Lengend Snippet: a) Inhibition of with CK1, but not CK2, specific kinase inhibitors causes dephosphorylation of pS935 of wildtype (WT) and K1906M in a dose dependent manner. Representative blots of 3 independent experiments. b) Dose response curve of IC261 on phosphorylation of S935 quantified from 2a. IC50 for WT = ~176 µM, IC50 for K1906M = 152 µM. Graph shows mean +/− SEM (n=3). c) CK1α inhibition with 200 µM IC261 abolishes LRRK2/14-3-3 interaction. Representative blots from 4 independent experiments. d) Quantitation of blots in 2c. Graph shows mean +/− SEM (n=3). Statistical significance tested with two-way ANOVA with Bonferroni post-hoc test (** p<0.01; ****p<0.0001). e) CK1α inhibition with IC261 in 14 DIV neurons showed a dose-dependent decrease of endogenous pS935. Representative blots from 3 independent experiments. f) Quantitation of blots in 2e. Graph shows mean +/− SEM. g) Experimental overview of acute brain ex vivo experiment. Coronal brain slices of 1mm thickness were prepared, and slices from one half of the brain from the same animal were treated with DMSO and the other half with either IC261 (#1) or LRRK-IN1 (#2). h) Adult non-transgenic wildtype mouse (4–9 weeks) acute brain ex vivo slices treated with DMSO, 1 µM LRRK2-IN1 and 300 µM IC261 for 2 hrs. S935 phosphorylation was reduced upon treatment with LRRK2-IN1 and IC261. i) Quantitation of blots in 2h. Graph shows mean +/− SEM (n=7). Statistical significance tested with paired t-test comparing treatments within a group (* p<0.05; **p<0.01).

Article Snippet: Equal amounts of immunopurified LRRK2 per experiment were incubated with 100 μM non-radioactive ATP, 100 ng recombinant active CK1 isoforms (Signal Chem Cat# C64-10G, C65-10G, C66-10G, C68-11G, C68-10BG, C68-10CG) or WEE1 (Active Motif Cat# 31201) and with or without DMSO, LRRK2-IN1 and IC261, in a final volume of 20 μl in cold kinase buffer.

Techniques: Inhibition, De-Phosphorylation Assay, Phospho-proteomics, Quantitation Assay, Ex Vivo, Transgenic Assay

Journal: Nature communications

Article Title: Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7

doi: 10.1038/ncomms6827

Figure Lengend Snippet: a) LRRK2 is a substrate of CK1α. CK1α phosphorylates LRRK2, in vitro , at S910, S935, S955 and S973 sites. Representative blots of 3 independent experiments. b) Consensus sequence of CK1 phosphorylation. The S/Tp-X-X-S/T is the canonical phosphorylation motif . Alternative phosphorylation motif of CK1 consist of an SLS motif followed by an acidic cluster in positions n+7 (underlined ). Sequence analysis of LRRK2 shows that serines 910, 935 and other constitutively phosphorylated serines at 973/975/976 is a weak consensus site for canonical and non-canonical CK1α phosphorylation. Phosphorylated serines of LRRK2 are shown in red. c) IC261 but not LRRK2-IN1, inhibited CK1α phosphorylation of LRRK2 in vitro . Concentrations of inhibitors used were 50 mM IC261 and 100 nM LRRK2-IN1. Results were consistent even when higher concentration of inhibitor, 100 mM IC261 and 1 µM LRRK2-IN1, was used . Representative blots of 3 independent experiments. d) Quantitation of blots in 3c. Graph shows mean +/− SEM. Statistical significance tested with two-way ANOVA with Bonferroni post-hoc test (* p<0.05; ** p<0.01; n.s. = not significant). e) LRRK2 is dephosphorylated at S910, S935, S955 and S973 upon knockdown with CSNK1A1 siRNA in a LRRK2-kinase independent manner, as both WT and K1906M is dephosphorylated to the same extent. f) CSNK1A1 siRNA knockdown samples described in 3e were subjected to LC-MS/MS analysis for phospho-peptide mapping. The XIC peak area extracted from the LC-MS/MS data was used to calculate the relative abundance of the detected phospho-peptide in different conditions. Graph shows the quantitative loss of ~70–80%, of pS908, pS910, pS935, pS955, pS973 and pS976 from CSNK1A1 compared to NTC siRNA samples for both WT (filled circles) and K1906M (open circles).

Article Snippet: Equal amounts of immunopurified LRRK2 per experiment were incubated with 100 μM non-radioactive ATP, 100 ng recombinant active CK1 isoforms (Signal Chem Cat# C64-10G, C65-10G, C66-10G, C68-11G, C68-10BG, C68-10CG) or WEE1 (Active Motif Cat# 31201) and with or without DMSO, LRRK2-IN1 and IC261, in a final volume of 20 μl in cold kinase buffer.

Techniques: In Vitro, Sequencing, Phospho-proteomics, Concentration Assay, Quantitation Assay, Knockdown, Liquid Chromatography with Mass Spectroscopy

Journal: Nature communications

Article Title: Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7

doi: 10.1038/ncomms6827

Figure Lengend Snippet: a) CK1α and ARHGEF7 are binding partners of LRRK2. When pS935 is lost, either due to 1 µM LRRK2-IN1 or 200 µM IC261 treatments, CK1α interaction with LRRK2 is diminished. Association of ARHGEF7 with LRRK2 (WT, S910A/S935A and K1906M) was increased upon IC261 treatment but no change in LRRK2-ARHGEF7 association occurred after LRRK2-IN1 treatment. Representative blots from 3 independent experiments. b) Quantitation of blots in 4a. Graph shows mean +/− SEM. Statistical significance tested with one-way ANOVA with Tukey’s post-hoc test comparing treatments within a group (* p<0.05; ** p<0.01; *** p<0.001; n.s. = not significant).

Article Snippet: Equal amounts of immunopurified LRRK2 per experiment were incubated with 100 μM non-radioactive ATP, 100 ng recombinant active CK1 isoforms (Signal Chem Cat# C64-10G, C65-10G, C66-10G, C68-11G, C68-10BG, C68-10CG) or WEE1 (Active Motif Cat# 31201) and with or without DMSO, LRRK2-IN1 and IC261, in a final volume of 20 μl in cold kinase buffer.

Techniques: Binding Assay, Quantitation Assay

Journal: Nature communications

Article Title: Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7

doi: 10.1038/ncomms6827

Figure Lengend Snippet: a) 293FT cells were co-expressed with FLAG-tagged LRRK2 and either with myc-tagged GUS (negative control for transfection), Rab7L1 WT or Q67L (loss of function variant), and also transfected with siRNA against Rab7L1 , ARHGEF7 or CSNK1A1 . Cells were stained for FLAG (green), TGN46 (red), myc (yellow), and nucleus (blue). The assay was setup in a 96-well plate format and the Golgi phenotype was imaged on a Cellomics VTI arrayscan. An automated counting of cells with LRRK2/TGN46-positve Golgi clustering was performed using the spot detector bioapplication of the Cellomics software to avoid operator bias. Scale bar shown is 50µm. b) Quantitation of proportion of cells with Golgi clustering phenotype shown in 6a. Data is from 3 independent experiments with 6 wells per condition and 1000 cells were counted per well. Graph shows mean ± SEM. Statistical significance tested with two-way ANOVA with Tukey’s post-hoc test comparing test siRNA to NTC within a group (**p<0.01; *** p<0.001; ****p<0.0001).

Article Snippet: Equal amounts of immunopurified LRRK2 per experiment were incubated with 100 μM non-radioactive ATP, 100 ng recombinant active CK1 isoforms (Signal Chem Cat# C64-10G, C65-10G, C66-10G, C68-11G, C68-10BG, C68-10CG) or WEE1 (Active Motif Cat# 31201) and with or without DMSO, LRRK2-IN1 and IC261, in a final volume of 20 μl in cold kinase buffer.

Techniques: Negative Control, Transfection, Variant Assay, Staining, Software, Quantitation Assay

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Antifibrotic effects of specific targeting of the 5‐hydroxytryptamine 2B receptor (5‐HT 2B R) in murine models and ex vivo models of scleroderma skin

doi: 10.1002/art.43151

Figure Lengend Snippet: Inhibition of 5‐HT 2B R by AM1476 modulates the expression of MAOB, SCL6A4, CCL2, CCL5, PAI‐1, and P‐STAT3 in murine cGvHD and PCS slices of SSc skin. (A and B) Murine cGvHD model. (A) Representative images of immunofluorescence staining and (B) quantification of Maob, Slc6a4, Ccl2, CCl5, Pai‐1, and P‐Stat3 in the skin of syngeneically and allogeneically transplanted mice with or without AM1476 treatment. All data are presented as mean ± SEM, with individual values displayed as column plus dots. Differences between the groups were tested for their statistical significance by one‐way analysis of variance with Dunnett's multiple comparison. Adjusted P values less than 0.05 were considered significant. Adjusted P values are expressed as follows: *0.05 > P > 0.01; **0.01 > P > 0.001; ***0.001 > P > 0.0001; **** P < 0.0001 as compared to the control allogenic group. (C and D) PCS slices of SSc skin. (C) Representative immunofluorescence staining images and (D) quantification of MAOB, SLC6A4, CCL2, CCL18, PAI‐1, and P‐STAT3 in SSc‐PCS with or without AM1476. All data points are presented as individual values displayed as dots. Differences between the groups were tested for their statistical significance by two‐way analysis of variance with Sidak's multiple comparison. Adjusted P values less than 0.05 were considered significant. Adjusted P values are expressed as follows: *0.05 > P > 0.01; **0.01 > P > 0.001; ***0.001 > P > 0.0001; **** P < 0.0001 as compared to the SSc‐PCS control group. 5‐HT 2B R, 5‐hydroxytryptamine 2B receptor; cGvHD, chronic graft‐versus‐host disease; PCS, precision cut skin; SSc, systemic sclerosis. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.43151/abstract .

Article Snippet: The staining of skin sections was performed by using the antibodies against CCL2 (LS‐C169178‐100, LSBio, 1:100 dilution), CCL5 (MAB478‐100, Biotechne, 1:100 dilution), CCL18 (22303‐1‐AP, Proteintech, 1:100 dilution), PAI‐1 (66261‐1‐Ig, Proteintech, 1:100 dilution), P‐STAT3 (MA5‐15193, Thermo Scientific, 1:100 dilution), MAOB (12602‐1‐AP, Proteintech, 1:100 dilution), or SLC6A4 (LS‐C154958‐100, LSBio, 1:100 dilution).

Techniques: Inhibition, Expressing, Immunofluorescence, Staining, Comparison, Control

Journal: PLoS ONE

Article Title: Identification of a Tumor Specific, Active-Site Mutation in Casein Kinase 1α by Chemical Proteomics

doi: 10.1371/journal.pone.0152934

Figure Lengend Snippet: A) Amino acid sequence of the wild type peptide. B) LC-MS/MS spectra for the CSNK1A1 peptide. The colon tumor sample is off-set in the Y-axis by 50 units for clarity and the region of interest (ROI) is highlighted. C) Region of interest in the LC-MS/MS spectra for colon tumor and matched control samples. Each spectrum is normalized to the highest intensity ion. D) Extracted ion chromatograms for CSNK1A1 from both colon tumor and matched control samples. Signals are normalized to the peak eluting at 59.8 minutes.

Article Snippet: An expression plasmid encoding DDK-tagged human CSNK1A1 was obtained from Origene (catalogue number RC217936, corresponding to NCBI reference sequence NP_001020276.1, splice form 1).

Techniques: Sequencing, Liquid Chromatography with Mass Spectroscopy, Control

Journal: PLoS ONE

Article Title: Identification of a Tumor Specific, Active-Site Mutation in Casein Kinase 1α by Chemical Proteomics

doi: 10.1371/journal.pone.0152934

Figure Lengend Snippet: A) Amino acid sequence of the mutant peptide. B) LC-MS/MS spectra for wild-type and mutant protein. The mutant sample is off-set by 50 units for clarity and the region of interest is highlighted. C) Region of interest in the LC-MS/MS spectra for colon tumor and matched control samples. Each spectrum is normalized to the highest intensity ion. D) Extracted ion chromatograms from HEK293 lysates transfected with either wild-type or mutant CSNK1A1. The wild-type sample is normalized to the peak eluting at 63.0 minutes and the mutant sample is normalized to the peak eluting at 59.8 minutes.

Article Snippet: An expression plasmid encoding DDK-tagged human CSNK1A1 was obtained from Origene (catalogue number RC217936, corresponding to NCBI reference sequence NP_001020276.1, splice form 1).

Techniques: Sequencing, Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Control, Transfection

Journal: PLoS ONE

Article Title: Identification of a Tumor Specific, Active-Site Mutation in Casein Kinase 1α by Chemical Proteomics

doi: 10.1371/journal.pone.0152934

Figure Lengend Snippet: HEK293 cells were transfected with either no DNA (Mock), CSNK1A1 wild type or D136N plasmids (CK1-WT and CK1-M, respectively).

Article Snippet: An expression plasmid encoding DDK-tagged human CSNK1A1 was obtained from Origene (catalogue number RC217936, corresponding to NCBI reference sequence NP_001020276.1, splice form 1).

Techniques: Transfection

Journal: PLoS ONE

Article Title: Identification of a Tumor Specific, Active-Site Mutation in Casein Kinase 1α by Chemical Proteomics

doi: 10.1371/journal.pone.0152934

Figure Lengend Snippet: A) A representative clone showing the mutated nucleotide (*). B) Alignment of all 41 sequences. Differences between pseudogene and CSNK1A1 are indicated by a plus symbol.

Article Snippet: An expression plasmid encoding DDK-tagged human CSNK1A1 was obtained from Origene (catalogue number RC217936, corresponding to NCBI reference sequence NP_001020276.1, splice form 1).

Techniques:

Journal: EMBO Molecular Medicine

Article Title: Casein kinase I epsilon interacts with mitochondrial proteins for the growth and survival of human ovarian cancer cells

doi: 10.1002/emmm.201101094

Figure Lengend Snippet: Representative figures of immunohistochemical staining of normal ovaries and ovarian tumour tissues for CKIε using the monoclonal mouse anti-human CKIε antibody from BD Biosciences. Statistical analysis of CKIε staining according to diagnostic and histological characteristics. Western blot analysis of CKIε expression in normal HOSE cells and ovarian cancer cell lines. Quantified CKIε signals presented at the bottom were normalized to signals of the β-actin of the cell lines. From left to right: HOSE 1-15, HOSE624, HOSE80-PC, OVCA810, RMG1, OVCA420, SKOV3, MCAS, DOV13, OVCA680, TOV21G, TOV112D, RMUGS, RMUGL, OVCA3, OVCA429, OVCA432, OVCA433, SKOV3-IP and HeyA8.

Article Snippet: For the introduction of CKIε into HOSE cells, full-length CKIε cDNA expression construct and empty vector were purchased from OriGene Technologies (Rockville, MD).

Techniques: Immunohistochemical staining, Staining, Diagnostic Assay, Western Blot, Expressing

Journal: EMBO Molecular Medicine

Article Title: Casein kinase I epsilon interacts with mitochondrial proteins for the growth and survival of human ovarian cancer cells

doi: 10.1002/emmm.201101094

Figure Lengend Snippet: Western blot analysis using the monoclonal mouse anti-human CKIε from BD Biosciences to show the increased expression of CKIε in the ectopic expression clones compared with the vector transfected clones (left). The growth rates of the four HOSE sublines determined by cell counting (right). Micrographs of ectopic expression clones and vector transfected clones growing in Matrigel culture.

Article Snippet: For the introduction of CKIε into HOSE cells, full-length CKIε cDNA expression construct and empty vector were purchased from OriGene Technologies (Rockville, MD).

Techniques: Western Blot, Expressing, Clone Assay, Plasmid Preparation, Transfection, Cell Counting

Journal: EMBO Molecular Medicine

Article Title: Casein kinase I epsilon interacts with mitochondrial proteins for the growth and survival of human ovarian cancer cells

doi: 10.1002/emmm.201101094

Figure Lengend Snippet: MTT assays to investigate the effects of CKIε/δ inhibitors IC261, PF-670462 and PF-4800567 on the proliferation rate of ovarian cancer cell lines. The p -values of differences between treated and untreated cells are <0.05 between 10 and 50 µM for IC261, between 20 and 50 µM for PF-670462 and PF-4800567, respectively. Western blot analysis to show the expression levels of CKIε and β-catenin in the ovarian cancer cells harbouring different shRNA constructs. The antibodies were from BD Biosciences. The ovarian cancer cells harbouring, respectively, CKIε shRNA constructs 3 and 4 demonstrated reduced proliferation rates. Migration rates of ovarian cancer cells harbouring control shRNA, CKIε shRNA construct 3 and CKIε shRNA construct 4 measured by transwell assay. Representative figures of the migrated cells on the membranes are shown on the right. Tumour burden as measured by whole body luminescence imaging (left) and excised tumour weights (right). The inter-quarter ranges (IQR) for the dispersion of excised tumour weights were 1.301 for the Control shRNA group and 0.878 for the CKIε shRNA group.

Article Snippet: For the introduction of CKIε into HOSE cells, full-length CKIε cDNA expression construct and empty vector were purchased from OriGene Technologies (Rockville, MD).

Techniques: Western Blot, Expressing, shRNA, Construct, Migration, Control, Transwell Assay, Imaging, Dispersion

Journal: EMBO Molecular Medicine

Article Title: Casein kinase I epsilon interacts with mitochondrial proteins for the growth and survival of human ovarian cancer cells

doi: 10.1002/emmm.201101094

Figure Lengend Snippet: Reporter luciferase assay to evaluate any changes in the β-catenin activity of the MCAS ovarian cancer cells harbouring control and CKIε knockdown constructs as described in Materials and Methods Section. The β-catenin binding elements-harbouring luciferase construct ( pTOPFLASH ) and control luciferase construct ( pFOPFLASH ) used in the transfections are indicated at the bottom. * p = 0.519; ** p = 0.734. Colloidal Coomassie universal protein dye-stained gel to show the resolved immunoprecipitated proteins using CKIε antibody (BD Biosciences) and a non-specific mouse control antibody. Lanes of two separate co-immunoprecipitation reactions are shown here. Open block arrows indicate the protein bands that were not present in the control lanes. The heavy chains and light chains of the immunoprecipitation antibodies are marked by solid block arrows. Confocal immunofluorescence of wild-type MCAS cells to demonstrate the co-localization of CKIε and mitochondrial proteins. The fluorescence of mitochondrial proteins were pseudo-coloured as green, whereas the fluorescence of CKIε was pseudo-coloured as red. COX IV was used as marker for intracellular mitochondria. Western blot analysis to show the expression of the three mitochondrial proteins in the control and knockdown cell lines. Measurement of intracellular ATP content in the control and CKIε knockdown cell lines.

Article Snippet: For the introduction of CKIε into HOSE cells, full-length CKIε cDNA expression construct and empty vector were purchased from OriGene Technologies (Rockville, MD).

Techniques: Luciferase, Activity Assay, Control, Knockdown, Construct, Binding Assay, Transfection, Staining, Immunoprecipitation, Blocking Assay, Immunofluorescence, Fluorescence, Marker, Western Blot, Expressing

Journal: EMBO Molecular Medicine

Article Title: Casein kinase I epsilon interacts with mitochondrial proteins for the growth and survival of human ovarian cancer cells

doi: 10.1002/emmm.201101094

Figure Lengend Snippet: The response of control and CKIε knockdown SKOV3 cells to Taxol and carboplatin as measured by MTT assays. Kaplan–Meier plots of the survivals of CKIε-positive and CKIε-negative tumours. Univariate and multivariate analyses of the CKIε positivity together with other clinicopathological parameters in predicting clinical outcome.

Article Snippet: For the introduction of CKIε into HOSE cells, full-length CKIε cDNA expression construct and empty vector were purchased from OriGene Technologies (Rockville, MD).

Techniques: Control, Knockdown