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.

Fig 3: PA28γ interacts with E4F1, which can be enhanced by PA28γ-T23 phosphorylation.a Immunofluorescence analyses of the cellular localization of endogenous PA28γ and E4F1 in HNSCC cells. DAPI (blue), nucleus; Scale bars, 10 μm. b WCLs derived from HNSCC cells were immunoprecipitated with control IgG, anti-PA28γ, or anti-E4F1 antibody, followed by IB analysis of co-IP products and WCLs. c Schematic representation of the various E4F1 truncations used in the PA28γ-binding assays tested for their capacity to bind PA28γ. The ubiquitin E3 ligase active region is represented by a green box, while the C2H2 and C2HC zinc finger motifs are highlighted in yellow and pink, respectively (Left). IB analysis of co-IP products and WCLs derived from HEK293T cells transfected with Flag-PA28γ and Myc-E4F1 truncations (Right). d IB analysis of co-IP products and WCLs derived from PA28γ-rescued HEK293T cells transfected with indicated constructs. e PA28γ-rescued HEK293T cells transfected with the indicated plasmids were stimulated without or with 50 μM TBB for 30 min before harvesting. WCLs were immunoprecipitated with Flag-beads, followed by IB analysis of co-IP products and WCLs. f HSC-3 cells transduced with control or Flag-PA28γ lentiviral vectors and silenced with siNC or siCK2 were stimulated without or with 100ngml–1 EGF for 30 min before harvesting. WCLs were immunoprecipitated with Flag-beads, followed by IB analysis of co-IP products and WCLs. g IB analysis of co-IP products and WCLs derived from PA28γ-rescued UM1 cells treated with 100ngml–1 EGF for the indicated time before harvesting. h, i HSC-3 cells were stimulated with 50 μM TBB (h) or 1 μM or 5 μM CX-4945 (i), and 100ngml–1 EGF before harvesting. WCLs were immunoprecipitated with anti-PA28γ antibody, followed by IB analysis of co-IP products and WCLs. j PA28γ-rescued HSC-3 cells were stimulated without or with 50 μM TBB and 100ngml–1 EGF for 30 min before harvesting. WCLs were immunoprecipitated with Flag-beads, followed by IB analysis of co-IP products and WCLs. k HEK293T cells were transfected with indicated plasmids and treated without or with 100ngml–1 EGF and 50 μM TBB for 30 min before being subjected to GST-PD assays and IB analyses. Source data are provided as a Source Data file.

Fig 4: Phosphorylation of PA28γ-T23 reduces E4F1 protein levels, relieving its transcriptional activity.a Heatmap of 26 up-regulated and 13 down-regulated proteins from proteomic analysis between PA28γ WT and PA28γ-T23A-rescued HEK293T cells (both groups with three biological replicates). b Kaplan-Meier survival analysis of overall survival in HNSCC patients from the TCGA database based on E4F1 expression using Kaplan-Meier Plotter. Statistical significance was assessed by the log-rank (Mantel-Cox) test without adjustment for multiple comparisons. c Representative multiplex IHC images of tongue mucosal tissues from WT, PA28γT23A, and PA28γT23D mice with the indicated antibodies. Scale bars, 50 μm. d PA28γ WT, T23A or T23D were re-expressed in endogenous PA28γ-knockdown HNSCC cells to establish PA28γ WT, PA28γ-T23A, and PA28γ-T23D-rescued HNSCC cell lines. IB analysis of WCLs derived from HNSCC cells transduced with lentiviral vectors expressing shRNA against endogenous PA28γ and EV, Flag-PA28γ WT, T23A, or T23D. The upper band is exogenous Flag-PA28γ, the lower band is endogenous PA28γ as marked. O.E., overexpression; Endo., endogenous. e IB analysis of WCLs derived from the indicated HNSCC cells. f, g IB analysis of WCLs derived from PA28γ WT, PA28γ-T23D (f), or PA28γ-T23A-rescued (g) HEK293T cells transfected with the indicated plasmids. h, i IB analysis of WCLs derived from PA28γ-rescued HNSCC cells treated with 100ngml–1 EGF (h) or HSC-3 cells treated with 50 μM TBB (i) at indicated time points. Source data are provided as a Source Data file.

Fig 5: The PA28γ-proteasome mediates E4F1 degradation.a IB analysis of WCLs derived from HEK293T cells transfected with His-E4F1 and increasing doses of Flag-PA28γ. b Quantitative real-time PCR analysis of the indicated genes in HEK293T cells transfected with EV and increasing doses of Flag-PA28γ. Endo., endogenous. c HSC-3 cells stably expressing EV or Flag-PA28γ were treated with 100 μgml−1 CHX for the indicated time before harvesting. Quantification of E4F1 levels relative to tubulin levels is shown. d, e HEK293T cells stably expressing EV or Flag-PA28γ were transfected with Myc-E4F1 truncations, and treated with 100 μgml−1 CHX for the indicated time before harvesting. Quantification of Myc-E4F1 levels relative to tubulin levels is shown. f HEK293T cells transfected with the indicated plasmids were treated without or with 10 μM MG132 for 6 h before harvesting, and WCLs were collected for IB analysis. g HEK293T cells stably expressing shRNA against endogenous PA28γ were transfected with the indicated plasmids, and WCLs were collected for IB analysis. h Purified PA28γ, E4F1 and 20S proteasome in the absence or presence of 100 nM proteasome inhibitor epoxomicin (Epox) were incubated as indicated for 45 min, followed by IB analysis. i Purified PA28γ WT, PA28γ-T23A, E4F1, p21 (as a positive control for E4F1) and 20S proteasome were incubated as indicated for 45 min, followed by IB analysis. Data in (b–e) represent the mean ± SD of three biological replicates; statistical significance was assessed by two-sided unpaired t-test. Source data are provided as a Source Data file.