Fig 1: CK2 interacts with and phosphorylates PA28γ at T23.a WCLs derived from HSC-3 cells were immunoprecipitated with control IgG, anti-PA28γ, or anti-CK2β antibody, followed by IB analysis of co-IP products and WCLs. b Immunofluorescence analyses of the cellular localization of endogenous PA28γ and CK2β in HNSCC cells. DAPI (blue), nucleus; Scale bars, 10 μm. c Schematic representation of the evolutionarily conserved PA28γ-T23 site in different species. Residues around the T23 site have a common characteristic of CK2 kinase substrates, namely the predominance of acidic amino acid residues (E/D) between n-4 and n + 7. α1–4 denotes helix-α1–4. d IB analysis of IP products and WCLs derived from HEK293T cells transfected with the indicated plasmids. e In vitro kinase assays were performed with recombinant His-PA28γ WT and T23A proteins purified from E. coli system as substrates and purified CK2 proteins containing CK2α and CK2β as the source of kinase. IB analysis of the indicated proteins in vitro kinase assay samples. f, g IB analysis of WCLs derived from HEK293T cells transfected with the subunits of CK2 (f) and HNSCC cells treated with 100ngml–1 EGF before harvesting (g). h, i HNSCC cells silenced with siRNA negative control (siNC) or siCK2 (h) and HSC-3 cells silenced with siNC or CK2α/α‘/β siRNA (i) were stimulated without or with 100ngml–1 EGF for 30 min before harvesting for IB analysis. j HEK293T cells transfected with the indicated plasmids were treated with 100ngml–1 EGF. WCLs were immunoprecipitated with the indicated antibodies, followed by IB analysis of co-IP products and WCLs. k In vitro kinase assays were performed with recombinant His-PA28γ proteins purified from E. coli system as substrates and purified CK2 proteins containing CK2α and CK2β as the source of kinase. 2 μM or 20 μM CK2 inhibitor TBB was added as indicated. l IB analysis of IP products and WCLs derived from HEK293T cells transfected with the indicated plasmids and silenced with siNC or #1, #2 NIP30 siRNA. m IB analysis of WCLs derived from HEK293T cells transfected with the indicated plasmids and silenced with siNC or #1, #2 NIP30 siRNA. Source data are provided as a Source Data file.
Fig 2: Expression pattern of the CK2-mediated PA28γ-T23 phosphorylation axis during HNSCC progression.a–c Multiplex IHC analyses of E4F1 in the TMA containing 81 HNSCC patients with paired tumors and adjacent tissues. Assessment of its levels in tumors and adjacent tissues (a), as well as in different AJCC stages (b). Survival analysis with Kaplan-Meier curves was performed on its high and low levels (c). Scale bars, 100 μm. d Correlation analyses among CK2β, PA28γ-pT23, E4F1, and Cyclin A2 were performed based on multiplex IHC staining scores from the HNSCC TMA. Representative tumor samples are shown. Scale bars, 100 μm. e Expression patterns of CK2β, PA28γ-pT23, E4F1, and Cyclin A2 in normal tissues, premalignant lesions, primary tumors, and metastatic lymph nodes were analyzed based on multiplex IHC staining scores. Representative samples are shown. Scale bars, 100 μm. f Expression levels of CK2β, PA28γ-pT23, E4F1, and Cyclin A2 in HNSCC tissues with or without lymph node metastasis. g Schematic illustration of this study. The study reveals the expression patterns of PA28γ-pT23 in cancers along with its clinical prognostic implications (Upper left panel), the sensitivity of T23 mutations to carcinogen-induced HNSCC in mice (Lower left panel), and the molecular mechanisms by which the CK2-mediated PA28γ-pT23 axis regulates cancer cell proliferation and growth (Right panel). Statistical significance was assessed by two-sided paired t-test in (a) unpaired t-test in (b, e and f) log-rank (Mantel-Cox) test in (c) and two-sided Pearson’s correlation test in (d). Source data are provided as a Source Data file.
Supplier Page from Sino Biological, Inc. for Human CSNK2A2 Gene ORF cDNA clone expression plasmid, N-His tag