Journal: Genes

Article Title: Haploinsufficiency as a Foreground Pathomechanism of Poirer-Bienvenu Syndrome and Novel Insights Underlying the Phenotypic Continuum of CSNK2B -Associated Disorders.

doi: 10.3390/genes14020250

Figure Lengend Snippet: Figure 10. Impact of CSNK2B mutations on CK2 subunits’ interaction and kinase activity. (A) HEK293 cells were transiently co-transfected with CSNK2A1-HA and the Myc-tagged CSNK2B wild type and mutants. Protein complexes were immunoprecipitated using an anti-HA antibody. Proteins were subjected to SDS–PAGE gel and immunoblotted with an anti-Myc antibody and an anti-HA antibody. Total cell lysates from co-transfected and non-transfected cells (mock) were probed with anti-Myc, anti-HA and anti-Hsp90 as a loading control. (B) Measure of the kinase activity of wild type (WT) and mutant (Leu39Arg) CK2beta proteins. CSNK2B wild-type and mutant constructs were overexpressed with the catalytic subunit CSNK2A1-HA, followed by immunoprecipitation with anti-Myc antibody and luminescent ADP-Glo kinase assay with (+) and without (−) CK2 specific substrate. (C) Histogram showing a significant decrease in the kinase activity of the mutant complex compared to the wild type. Experiments were repeated three times with independently prepared cell extracts. ** p < 0.01 calculated by Student’s t-test (two-tailed, homoscedastic).

Article Snippet: Human wild-type CSNK2B (NM_001320) and mutant (CSNK2B_Leu39Arg and CSNK2B_Met132LeufsTer110) cDNAs, as well as the CSNK2A1 (NM_001895), were cloned into a pCMV6-Entry vector (Origene, Rockville, MD, USA).

Techniques: Activity Assay, Transfection, Immunoprecipitation, SDS Page, Control, Mutagenesis, Construct, Kinase Assay, Two Tailed Test

Journal: Cell death discovery

Article Title: CK2α-mediated phosphorylation of GRP94 facilitates the metastatic cascade in triple-negative breast cancer.

doi: 10.1038/s41420-024-01956-x

Figure Lengend Snippet: Fig. 5 Benzamidine, a novel CK2α inhibitor, reduces LRP6 stability by suppressing GRP94 phosphorylation. a CK2α kinase activity was measured after treatment with vehicle, benzamidine (10 μM and 50 μM), or silmitasertib (20 μM). Cell lysates were used for in vitro kinase assay to detect the intensity of phosphorylated substrate. Data are presented as relative activity of vehicle-treated sample. *p < 0.05; **p < 0.01 (Student’s t test). b Thermal shift assay for CK2α was performed after addition of benzamidine (10 and 50 μM). Positive derivative (d(RFU)/dT) curves are shown for control and treated with benzamidine (10 and 50 μM). Midpoint temperatures of protein-unfolding transition (Tm) are presented as bars. Values are presented as mean ± SD of at least three independent measurements. *p < 0.05; ***p < 0.001. One-way ANOVA was followed by Dunnett’s multiple comparisons test. c MDA-MB231 cells were seeded into a 12-well plate and transfected with TOPflash/ FOPflash reporter plasmids. After 24 h, cells were treated with benzamidine (10 and 50 μM) or vehicle for an additional 24 h. β-catenin transcriptional activity was assessed by measuring luminescence. TOPflash values are presented as fold change from control cells. β-galactosidase activity was used as a normalization control. Bars represent mean ± SD. *p < 0.05 (Student’s t test). d Wnt signaling-related markers were checked in MDA-MB231 cells treated with benzamidine (10 and 50 μM) for 24 h. β-actin band was used as a normalization control. e MDA-MB231 cells were transfected with Myc-GRP94 and treated with increasing concentrations of benzamidine. Cell extracts were then subjected to immunoprecipitation with anti-Myc antibody and detected with anti-p-Ser antibody. f MDA-MB231 cells were treated with benzamidine (50 μM). Stability of GRP94 and LRP6 was measured after adding CHX for the indicated time course, followed by western blotting analysis. Results are representative of at least three independent experiments and data are presented as mean ± SD.

Article Snippet: Recombinant CK2α protein was obtained from Origene (TP760181, Rockville, MD, USA).

Techniques: Phospho-proteomics, Activity Assay, In Vitro, Kinase Assay, Thermal Shift Assay, Control, Transfection, Immunoprecipitation, Western Blot

Journal: Cell death discovery

Article Title: CK2α-mediated phosphorylation of GRP94 facilitates the metastatic cascade in triple-negative breast cancer.

doi: 10.1038/s41420-024-01956-x

Figure Lengend Snippet: Fig. 7 Schematic diagram illustrating the important role of CK2α- GRP94-LRP6 axis in TNBC metastasis. In TNBC cells, elevated CK2α phosphorylates GRP94 at serine 306 residue, leading to increased stability of the GRP94 protein. As an ER chaperone, GRP94 promotes the maturation of LRP6 protein, resulting in activation of Wnt signaling. Collectively, the activation and upregulation of CK2α and GRP94 synergistically promote the metastasis of TNBC cells through the Wnt signaling pathway. By targeting CK2α-mediated phosphor- ylation of GRP94 with benzamidine, metastasis of TNBC can be effectively controlled as a therapeutic strategy.

Article Snippet: Recombinant CK2α protein was obtained from Origene (TP760181, Rockville, MD, USA).

Techniques: Residue, Activation Assay