Journal: bioRxiv

Article Title: Phosphoproteomics of cellular mechanosensing reveals NFATC4 as a regulator of myofibroblast activity

doi: 10.1101/2023.02.13.528335

Figure Lengend Snippet: Pathway and upstream regulator analysis reveals stiffness regulated processes. A. Heat map shows enriched terms across gene lists of clusters 1–10 in main , colored by p-values. Enrichment analysis was performed with Metascape ( www.metascape.org ). B. The graphs show CK2A1 targets predicted by Ingenuity Pathway Analysis (IPA) and their phosphorylation status in the phosphoproteomic data set. Turquoise color shades indicate low phosphorylation and pink color shades indicate high phosphorylation. Doted lines indicate an indirect interaction and continuous lines specify a direct interaction from prior knowledge databases. C. Results of pathway enrichment analysis of the IPA CK2A1 target sites using Fisher exact test (FDR < 5%). D. Immunoblot probed with an antibody specific for proteins containing a pS/pTDXE motif, which represents a CK2 phosphorylation consensus sequence. GAPDH served as a loading control (n=3).

Article Snippet: Antibodies used were: Anti-GAPDH (1:1000, Cell Signaling #5174S), Anti-CK II alpha (1:1000, Abcam ab70774), Anti-Phospho-CK2 Substrate Motif [(pS/pT)DXE] (1:1000, Cell Signaling #8738), Anti-Phospho-Myosin Light Chain 2 (Thr18/Ser19) (1:2000, Cell Signaling #3674), Anti-Phospho-FoxO3a (S7) (1:1000, Cell Signaling 14724S), Anti-Phospho-Paxillin (S126) (1:1000, Life Technologies 441022G), Anti-Phospho-CBX3 (Ser93) (1:1000, Cell Signaling #2600), Anti-rabbit IgG, HRP-linked (1:20,000, Cell Signaling #7074) Anti-mouse IgG, HRP-linked (1:20,000, Cell Signaling #7074).

Techniques: Western Blot, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Control of TRPM3 Ion Channels by Protein Kinase CK2-Mediated Phosphorylation in Pancreatic β-Cells of the Line INS-1

doi: 10.3390/ijms222313133

Figure Lengend Snippet: CK2 phosphorylates TRPM3 channels. Immunoprecipitated proteins from HEK293 cells (1) and TRPM3α2-expressing HEK293 cells (2) were incubated in the presence (+) or absence (-) of added CK2 and separated by gel electrophoresis. ( A ) Autoradiograph (short exposure, ~2 h) of proteins that were immunoprecipitated using monoclonal anti-TRPM3 antibodies and phosphorylated in the presence of [γ- 32 P]ATP. ( B ) The same as in ( A ) but in the presence of [γ- 32 P]GTP and the absence of added CK2 (long exposure, ~24 h). ( C ) Same as in ( B ), but proteins were immunoprecipitated using polyclonal anti-TRPM3 antibodies. ( D ) Same as in ( C ), but separated proteins were blotted onto a PVDF membrane and analyzed with a phospho-CK2 substrate motif antibody.

Article Snippet: For immunoblot analysis of phosphorylated protein with a phospho-CK2 substrate motif antibody (Cell Signaling #8738), extraction, immunoprecipitation, phosphorylation using GTP, and SDS PAGE were performed as described above.

Techniques: Immunoprecipitation, Expressing, Incubation, Nucleic Acid Electrophoresis, Autoradiography

Journal: International Journal of Molecular Sciences

Article Title: Control of TRPM3 Ion Channels by Protein Kinase CK2-Mediated Phosphorylation in Pancreatic β-Cells of the Line INS-1

doi: 10.3390/ijms222313133

Figure Lengend Snippet: TRPM3-mediated Ca 2+ signals are inhibited by CK2. ( A ) Ca 2+ signals induced by PS/CIM0216 in stable TRPM3α2-overexpressing HEK293 cells (HEKα2) in the presence (red trace) or absence (black trace, solvent DMSO only) of the CK2 inhibitor CX-4945. Cells were pre-incubated for 30 min in CX-4945 or DMSO. ( B ) Fura-2 Ca 2+ imaging experiments with HEKα2 cells transfected with cDNA encoding EGFP–CK2α fusion proteins and stimulated with pregnenolone sulfate (PS)/CIM0216. Ca 2+ signals of green fluorescent cells (green) were compared to non-green cells (black) of the same dish as well as to non-transfected cells (blue). The number of independent experiments and the total number of analyzed cells are each indicated in brackets.

Article Snippet: For immunoblot analysis of phosphorylated protein with a phospho-CK2 substrate motif antibody (Cell Signaling #8738), extraction, immunoprecipitation, phosphorylation using GTP, and SDS PAGE were performed as described above.

Techniques: Incubation, Imaging, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Control of TRPM3 Ion Channels by Protein Kinase CK2-Mediated Phosphorylation in Pancreatic β-Cells of the Line INS-1

doi: 10.3390/ijms222313133

Figure Lengend Snippet: CK2 phosphorylation of a single serine residue of TRPM3. ( A ) Localization of putative CK2 phosphorylation sites within TRPM3 proteins. The mouse Trpm3 gene comprises 28 exons (upper panel, ). Alternative N -termini of α-isoforms and β-isoforms are encoded by exon 1 and exon 2, respectively (shown in red). Likewise, as a result of alternative splicing, the protein regions encoded by exons 8, 13b, 15, 17, and 24b (shown in red) are absent in several isoforms. The organization of domains of the encoded TRPM3 proteins (light gray bar) is shown below true to scale. Well-conserved domains such as transmembrane helices S1–S6 (dark gray), channel pore (P, violet) TRP domain (TRP, green) a N /C-terminal connecting helix (CH, blue), and a coiled coil domain (CC, orange) are indicated . The positions of 23 serine (S) and 9 threonine (T) amino acid residues (aa), representing putative CK2 phosphorylation sites, are indicated by numbers. ( B ) Analysis of all putative CK2 phosphorylation sites within TRPM3 reveals selective phosphorylation of S 1280 . In all, 15 mer-peptides each comprising one of the 32 putative phosphorylation sites (S/T XX D/E) within the complete TRPM3 amino acid sequence (including all known exons) were synthesized on cellulose membranes. Corresponding peptides carrying S/T to A mutations were synthesized next to them, each. Membranes were incubated with [γ- 32 P]ATP in the presence (+CK2) or absence (-CK2) of protein kinase CK2. Peptides of the sequence RRRDDDSDDD/RRRDDDADDD served as positive (con+) and negative (con-) controls, respectively. Note the selective phosphorylation of S 1280 (highlighted by a red rectangle). ( C ) S 1280 (highlighted in red, corresponding to S 1172 of TRPM3α2) is not conserved within TRPM proteins as shown by sequence alignment of mouse TRPM3 (NP_001030319.1) with its closest relatives TRPM1 (NP_001034193.2), TRPM6 (NP_700466.1), and TRPM7 (NP_067425.2). Amino acid residues identical to TRPM3 are highlighted by gray backgrounds. Amino acid residues belonging to the TRP-domain , the connecting helix , and the coiled coil domain are labeled in green, blue, and orange, respectively.

Article Snippet: For immunoblot analysis of phosphorylated protein with a phospho-CK2 substrate motif antibody (Cell Signaling #8738), extraction, immunoprecipitation, phosphorylation using GTP, and SDS PAGE were performed as described above.

Techniques: Sequencing, Synthesized, Incubation, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Control of TRPM3 Ion Channels by Protein Kinase CK2-Mediated Phosphorylation in Pancreatic β-Cells of the Line INS-1

doi: 10.3390/ijms222313133

Figure Lengend Snippet: Protein kinase CK2 controls TRPM3-mediated Ca 2+ signals in INS-1 β-cells. ( A ) The pregnenolone sulfate (PS)/CIM0216-induced Ca 2+ entry in INS-1 cells was exclusively induced by TRPM3 and increased after treatment with the CK2 inhibitor, CX-4945. Cells from three independent TRPM3-deficient INS-1 cell lines (green, blue, and violet traces) did not respond to the TRPM3 agonists PS (100 µM) and CIM0216 (1 µM). In contrast, wild-type INS-1 cells that were pre-incubated for 30 min in 10 µM CX-4945 (red trace) showed increased PS/CIM0216-induced Ca 2+ signals compared to that in control cells treated with solvent (DMSO) only (black trace). ( B ) Pregnenolone sulfate (PS)/CIM0216-induced Ca 2+ signals in INS-1 cells were reduced after the introduction of recombinant CK2α. INS-1 cells were transfected with pEGFP-CK2α. Green fluorescent cells expressing EGFP–CK2α fusion proteins (green trace) were compared to non-green cells of the same dish (black trace). The number of independent experiments and the total number of analyzed cells are indicated in brackets, each.

Article Snippet: For immunoblot analysis of phosphorylated protein with a phospho-CK2 substrate motif antibody (Cell Signaling #8738), extraction, immunoprecipitation, phosphorylation using GTP, and SDS PAGE were performed as described above.

Techniques: Incubation, Recombinant, Transfection, Expressing

Journal: Communications Biology

Article Title: Elevating CDCA3 levels in non-small cell lung cancer enhances sensitivity to platinum-based chemotherapy

doi: 10.1038/s42003-021-02136-8

Figure Lengend Snippet: a Beeswarm plot showing CDCA3 levels assessed in control or CK2 depleted H460 cells that were treated with or without cisplatin. CDCA3 levels determined by analysis of high throughput immunofluorescence microscopy images. Data points represent CDCA3 levels within individual cells from a minimum of 1200 cells (see Supplementary Fig. ). Horizontal lines indicate median values (unpaired Student’s t test, **** P = < 0.0001). b Densitometry quantification of western blot analysis in Supplementary Fig. , with dot points representing average log2 of relative endogenous CDCA3 levels from NSCLC cell lines treated with or without CX-4945. Representative of three independent experiments with lines connecting respective untreated and cisplatin treated cell lines (paired Student’s t test, * P = 0.0126). c Immunoprecipitation analysis of endogenous CDCA3 to assess the CK2-mediated phosphorylation of CDCA3 in untreated or cisplatin treated H460 cells. d Immunoprecipitation analysis of ectopic CDCA3 from H460 cell lysates to assess the impact of cisplatin, CX-4945 or combination of both upon CK2-mediated phosphorylation of CDCA3. e Immunoprecipitation analysis to assess CK2-mediated phosphorylation of enriched endogenous CDCA3 from nocodazole arrested H460 cells in prometaphase (prometa.) or H460 cells arrested at the G1/S cell cycle boundary by double thymidine block. c – e CK2 phosphorylation assessed using antibody specific for CK2 phosphorylation motifs within substrates. f Immunoprecipitation analysis of ectopic HA-Cdh1 with ectopic wild-type CDCA3-FLAG from H460 cells treated with cisplatin, CX-4945 or a combination of both. All in vitro experiments are representative of three independent repeats.

Article Snippet: Antibodies against the HA tag (#3724) and phospho-CK2 substrate motif (#8738) were purchased from Cell Signaling Technology (Genesearch, Australia).

Techniques: High Throughput Screening Assay, Immunofluorescence, Microscopy, Western Blot, Immunoprecipitation, Blocking Assay, In Vitro

Journal: Science signaling

Article Title: Dual phosphorylation of Ric-8A enhances its ability to mediate G protein α subunit folding and to stimulate guanine nucleotide exchange

doi: 10.1126/scisignal.aap8113

Figure Lengend Snippet: (A) GPS 3.0 was used to evaluate sites within Ric-8A with potential for CK2-mediated phosphorylation (47). The prediction was run at the high-stringency setting using a maximal false-positive rate threshold cutoff of 2%. Higher relative scores represent greater potential for phosphorylation. (B) Phosphosite identification in rat Ric-8A purified from insect cells and from E. coli-produced Ric-8A treated with CK2 was made by MS analysis of peptides from tryptic and chymotryptic digests. Red boxed residues were phosphorylated sites in insect cell–produced Ric-8A, yellow boxed residues were phosphorylated sites in E. coli–produced Ric-8A treated with CK2, and blue boxed residues were phosphorylated sites obtained for both proteins. All phosphosites were identified by Mascot software and confirmed by analysis of CID spectra. Peptide coverage of the three C-terminal phosphosites (Ser522, Ser523, and Ser527) was not attained for insect cell–produced Ric-8A. The 10-residue acidic sequence that contains the two consecutive CK2 sites of Ser435 and Thr440 is underlined. (C) The 10-residue acidic sequence is highly conserved across vertebrate Ric-8 proteins. The phosphosites corresponding to rat Ric-8A Ser435 and Thr440 are invariantly serine or threonine residues in all homologs examined. Dictyostelium Ric-8 is a shortened protein that lacks the acidic sequence stretch altogether. (D) Insect cell–produced rat wild-type (WT), S435A, and T440A Ric-8A proteins were treated with or without CK2 and then analyzed by Western blotting with an antibody specific for the CK2 consensus site (pS/pT)-D-X-E, which recognizes the phosphorylated Thr440 site (p-Thr440), the 6383 Ric-8A antiserum that recognizes the phosphorylated Ser435 site (p-Ser435), and the 1184 antiserum that recognizes Ric-8A. (E) Insect cell–produced mouse Ric-8B was treated with and without alkaline phosphatase and analyzed by Western blotting with the Ric-8A phosphosite-specific antibodies that also detect mouse Ric-8B p-Ser468 and p-Ser472, respectively. Ric-8B antiserum 2413 was used to detect the Ric-8B alkaline phosphatase–dependent gel shift after protein resolution by Phos-tag PAGE. (F) Purified Ric-8A from insect cells was treated with or without alkaline phosphatase, whereas E. coli–produced Ric-8A was treated with or without CK2. The proteins were analyzed by Western blotting with the p-Thr440, p-Ser435, and 1184 antibodies. (G) Insect–produced WT Ric-8A and the indicated Ric-8A alanine substitution mutant proteins produced in E. coli were treated with or without CK2 and analyzed by Western blotting with the p-Thr440 and 1184 Ric-8A antibodies. The proteins were also resolved by Phos-tag PAGE and analyzed by Western blotting with the 1184 Ric-8A antibody, as indicated. (H) WT, T440A, and S435A/T440A Ric-8A-ΔCT purified from E. coli were treated with or without CK2, resolved by SDS-PAGE and Phos-tag PAGE, and analyzed by Western blotting with the p-Thr440, p-Ser435, and 1184 Ric-8A antibodies, as indicated. Data in (D) to (H) are representative of more than three independent experiments.

Article Snippet: Monoclonal rabbit phospho-CK2 motif (pS/pT)-D-X-E antibody (Cell Signaling Technology) was found to specifically recognize phosphorylation at site Thr 440 of recombinant rat Ric-8A ( pT -D-T-E) but failed to recognize human Ric-8A ( pT -D-T-D).

Techniques: Purification, Produced, Software, Sequencing, Western Blot, Electrophoretic Mobility Shift Assay, Mutagenesis, SDS Page