Journal: bioRxiv

Article Title: diaPASEF-Powered Chemoproteomics Enables Deep Kinome Interaction Profiling

doi: 10.1101/2024.11.22.624841

Figure Lengend Snippet: Utilizing 2 nd gen kiCCA to prioritize kinase targets for pharmacologically manipulating NB cell NMT states. (A) STRING interaction networks (v12.0) of kinase PPIs predicted by 2 nd gen kiCCA in the isogenic NB cell lines SK-N-SH (mesenchymal-like) and SH-SY5Y (noradrenergic neuronal-like). Only physical interactions were used to build the STRING network. Relates to Table S1 . (B) Results from GSEA of global protein expression data, comparing SH-SY5Y cells treated with either the CK2 inhibitor SGC-CK2-1 or DMSO (vehicle) for 4 days. Only pathway terms that achieved an FDR < 0.05 are shown. Relates to Table S2 . (C) Volcano plot showing expression differences of neuronal marker proteins between SH-SY5Y cells treated with either the CK2 inhibitor SGC-CK2-1 or dimethyl sulfoxide (DMSO, vehicle) for 4 days. Proteins included in the GOBP gene set ‘Neuronal Differentiation’ were used to define proteins as neuronal marker proteins. Statistics: two sample Student’s t-test, BH-FDR < 0.05, N = 3 Relates to Table S2 . (D) Results from GSEA of global protein expression data, comparing SK-N-SH cells treated with either the TBK1 inhibitor GSK8612 or DMSO (vehicle) for 4 days. Only pathway terms that showed an FDR < 0.05 are shown. Relates to Table S2 . (E) Differences in the expression of proteins that are the transcriptional targets of TGFβ-SMAD3, JNK-AP1, and NF-κB signaling between SK-N-SH cells treated with either the TBK1 inhibitor GSK8612 or DMSO (vehicle) for 4 days. Result of global proteome profiling. All proteins shown significantly differed in expression. Statistics: two sample Student’s t-test, BH-FDR < 0.05, N = 4. Refers to Table S2 . (F) Trans well migration assay showing that CK2 inhibition with SGC-CK2-1 promoted migration in both SH-SY5Y cells and SK-N-SH cells, and that TBK1 and tyrosine kinase inhibition using GSK8612 and dasatinib, respectively, significantly inhibited cell migration only in the SK-N-SH cell line. Statistics: two sample Student’s t-test p < 0.05, N = 4; error bars are the S.D.

Article Snippet: For global proteome profiling, 0.3x10 6 SK-N-SH or SH-SY5Y cells per well were seeded on 6-well plates and allowed to adhere for 24 h. Then, the TBK1 inhibitor GSK8612 (2 µM final, MCE), the CK2 inhibitor SGC-CK2- 1 (2 µM final, MCE), or DMSO (vehicle control) were added.

Techniques: Expressing, Marker, Migration, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Nerve injury augments Cacna2d1 transcription via CK2-mediated phosphorylation of the histone deacetylase HDAC2 in dorsal root ganglia

doi: 10.1016/j.jbc.2024.107848

Figure Lengend Snippet: Nerve injury augments the interaction between HDAC2 and CK2α in the DRG. A , representative immunoblotting images show that the anti-HDAC2 antibody precipitated CK2α, but not CK2β, in the DRG of rats subjected to spinal nerve ligation (SNL) or sham surgery. Three weeks after the surgery, total proteins from DRG tissues were extracted for immunoprecipitation (IP) with an anti-HDAC2 antibody. The immune complex was then subjected to immunoblotting for the detection of bound proteins. Normal rabbit IgG was used as a negative control in immunoprecipitation. B , quantification of the HDAC2-CK2α protein complex in the DRG from SNL and sham control rats (normalized to the HDAC2 protein band in IP samples, n = 6 rats per group). Data are expressed as means ± SD. ∗∗∗ p < 0.001 (two-tailed Student t test).

Article Snippet: The selective CK2 inhibitor, CX-4945 (#HY-50855; MedChemExpress, Monmouth Junction, NJ) or a vehicle consisting of 10% dimethyl sulfoxide and 90% saline mixed with sulfobutylether-β-cyclodextrin (#HY-17031, MedChemExpress) was administered intrathecally at a volume of 10 μl, followed by a 5 μl saline flush.

Techniques: Western Blot, Ligation, Immunoprecipitation, Negative Control, Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Nerve injury augments Cacna2d1 transcription via CK2-mediated phosphorylation of the histone deacetylase HDAC2 in dorsal root ganglia

doi: 10.1016/j.jbc.2024.107848

Figure Lengend Snippet: CK2 inhibition attenuates nerve injury-induced HDAC2 phosphorylation in the DRG. A and B , Representative immunoblot images ( A ) and quantification ( B ) show the protein levels of phospho-serine 394 of HDAC2 (pHDAC2 Ser394 ) and total HDAC2 in the DRG of rats subjected to spinal nerve ligation (SNL) or sham surgery (n = 6 rats per group). SNL and sham control rats were intrathecally injected with vehicle or CX-4945 (10 μg per day) for five consecutive days. Total proteins were extracted 2 h after the last injection. GAPDH was used as a loading control. Two-way ANOVA showed that there was a significant main effect for SNL (F (1, 20) = 36.9; p < 0.001) and CX-4945 treatment (F (1,20) = 63.9; p < 0.001) and a significant interaction between SNL and CX-4945 treatment (F (1, 20) = 5.19; p < 0.05). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (two-way ANOVA followed by Tukey’s post hoc test). Data are expressed as means ± SD.

Article Snippet: The selective CK2 inhibitor, CX-4945 (#HY-50855; MedChemExpress, Monmouth Junction, NJ) or a vehicle consisting of 10% dimethyl sulfoxide and 90% saline mixed with sulfobutylether-β-cyclodextrin (#HY-17031, MedChemExpress) was administered intrathecally at a volume of 10 μl, followed by a 5 μl saline flush.

Techniques: Inhibition, Western Blot, Ligation, Control, Injection

Journal: The Journal of Biological Chemistry

Article Title: Nerve injury augments Cacna2d1 transcription via CK2-mediated phosphorylation of the histone deacetylase HDAC2 in dorsal root ganglia

doi: 10.1016/j.jbc.2024.107848

Figure Lengend Snippet: CK2 inhibition attenuates nerve injury–induced pain hypersensitivity and α2δ-1 expression in the DRG. A , time course of changes in withdrawal thresholds tested with von Frey filaments, pressure, and thermal stimuli in sham and SNL rats intrathecally treated with vehicle or CX-4945 (10 μg per day) for five consecutive days (n = 8 rats per group). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, compared with SNL rats treated with vehicle at the same time point (two-way ANOVA with Tukey's post hoc test). B , quantitative PCR assays show the mRNA levels of Cacna2d1 in the DRG of sham and SNL rats intrathecally injected with vehicle or CX-4945 (10 μg per day) for five consecutive days (n = 8 rats per group). Two-way ANOVA showed that there was a significant main effect for SNL (F (1, 28) = 72.4; p < 0.001) and CX-4945 treatment (F (1,28) = 36.6; p < 0.001) and a significant interaction between SNL and CX-4945 treatment (F (1, 20) = 8.45; p < 0.01). ∗∗∗ p < 0.001 (two-way ANOVA with Tukey's post hoc test). C , representative immunoblotting images and quantification show the protein levels of α2δ-1 in the DRG of sham and SNL rats intrathecally injected with vehicle or CX-4945 for five consecutive days (n = 12 rats per group). Total proteins were extracted 2 h after the last injection. GAPDH was used as a loading control. Two-way ANOVA showed that there was a significant main effect for SNL (F (1, 44) = 130; p < 0.001) and CX-4945 treatment (F (1,44) = 30.9; p < 0.001) and a significant interaction between the SNL and CX-4945 treatment (F (1, 44) = 17.8; p < 0.001). ∗∗∗ p < 0.001 (two-way ANOVA with Tukey's post hoc test). Data are expressed as means ± SD.

Article Snippet: The selective CK2 inhibitor, CX-4945 (#HY-50855; MedChemExpress, Monmouth Junction, NJ) or a vehicle consisting of 10% dimethyl sulfoxide and 90% saline mixed with sulfobutylether-β-cyclodextrin (#HY-17031, MedChemExpress) was administered intrathecally at a volume of 10 μl, followed by a 5 μl saline flush.

Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Injection, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Nerve injury augments Cacna2d1 transcription via CK2-mediated phosphorylation of the histone deacetylase HDAC2 in dorsal root ganglia

doi: 10.1016/j.jbc.2024.107848

Figure Lengend Snippet: CK2 inhibition restores HDAC2 enrichment at the Cacna2d1 promoter in the injured DRG. A and B , quantification of the immunoprecipitated chromatin show the enrichment of HDAC2 proteins at the −21 to +74 bp region of the Cacna2d1 promoter ( A ) and in the +27 to +127 bp region of the Gapdh promoter ( B ) in the DRG. ChIP-qPCR was performed using the DRG tissues from sham and SNL rats intrathecally injected with vehicle or CX-4945 (10 μg per day) for five consecutive days (n = 6 rats per group). Two-way ANOVA showed that there was a significant main effect for SNL (F (1, 20) = 42.9; p < 0.001) and CX-4945 treatment (F (1,20) = 5.17; p < 0.01) and a significant interaction between SNL and CX-4945 treatment (F (1, 20) = 8.42; p < 0.01). ∗∗ p < 0.01, ∗∗∗ p < 0.001 (two-way ANOVA with Tukey's post hoc test). Data are expressed as means ± SD.

Article Snippet: The selective CK2 inhibitor, CX-4945 (#HY-50855; MedChemExpress, Monmouth Junction, NJ) or a vehicle consisting of 10% dimethyl sulfoxide and 90% saline mixed with sulfobutylether-β-cyclodextrin (#HY-17031, MedChemExpress) was administered intrathecally at a volume of 10 μl, followed by a 5 μl saline flush.

Techniques: Inhibition, Immunoprecipitation, Injection

Journal: The Journal of Biological Chemistry

Article Title: Nerve injury augments Cacna2d1 transcription via CK2-mediated phosphorylation of the histone deacetylase HDAC2 in dorsal root ganglia

doi: 10.1016/j.jbc.2024.107848

Figure Lengend Snippet: CK2 inhibition reduces histone acetylation at the Cacna2d1 promoter in the injured DRG. A – D , quantification of the immunoprecipitated chromatin show the enrichment of total H3ac ( A ), H4ac ( B ), H3K9ac ( C ), and H4K5ac ( D ) at the Cacna2d1 promoter (−21 to +74 bp). ChIP-qPCR was performed using the DRG tissues from sham and SNL rats intrathecally injected with vehicle or CX-4945 (10 μg per day) for five consecutive days (n = 6 rats per group). Two-way ANOVA showed that there was a significant interaction between SNL and CX-4945 treatment for the enrichment of H3ac (F (1, 20) = 34.4; p < 0.001), H4ac (F (1, 20) = 15.7; p < 0.001), and H3K9ac (F (1, 20) = 12.7; p < 0.01). ∗ p < 0.05, ∗∗∗ p < 0.001 (two-way ANOVA with Tukey's post hoc test). Data are expressed as means ± SD.

Article Snippet: The selective CK2 inhibitor, CX-4945 (#HY-50855; MedChemExpress, Monmouth Junction, NJ) or a vehicle consisting of 10% dimethyl sulfoxide and 90% saline mixed with sulfobutylether-β-cyclodextrin (#HY-17031, MedChemExpress) was administered intrathecally at a volume of 10 μl, followed by a 5 μl saline flush.

Techniques: Inhibition, Immunoprecipitation, Injection

Journal: The Journal of Biological Chemistry

Article Title: Nerve injury augments Cacna2d1 transcription via CK2-mediated phosphorylation of the histone deacetylase HDAC2 in dorsal root ganglia

doi: 10.1016/j.jbc.2024.107848

Figure Lengend Snippet: Schematic representation illustrates changes induced by nerve injury in CK2 and HDAC2 phosphorylation, histone acetylation at the Cacna2d1 promoter, and α2δ-1 expression in the DRG. Under normal conditions, CK2 minimally interacts with HDAC2, which is highly enriched by the Cacna2d1 gene promoter. Consequently, the acetylation levels of histones H3 and H4, alone with activating histone marks (H3K9ac or H4K5ac), are low at the Cacna2d1 promoter, keeping the Cacna2d1 transcription epigenetically repressed in the DRG. Nerve injury augments CK2 activity, leading to increased CK2-HDAC2 interactions, HDAC2 phosphorylation, and subsequent HDAC2 dissociation at the Cacna2d1 promoter in the DRG. The loss of HDAC2 at the Cacna2d1 promoter boosts histone acetylation levels and the presence of active histone marks, resulting in elevated Cacna2d1 transcription and α2δ-1 expression in the injured DRG.

Article Snippet: The selective CK2 inhibitor, CX-4945 (#HY-50855; MedChemExpress, Monmouth Junction, NJ) or a vehicle consisting of 10% dimethyl sulfoxide and 90% saline mixed with sulfobutylether-β-cyclodextrin (#HY-17031, MedChemExpress) was administered intrathecally at a volume of 10 μl, followed by a 5 μl saline flush.

Techniques: Expressing, Activity Assay