Fig 2: Mechanisms by which DYRK2 regulates the proteasome(a) DYRK2 does not affect 26S proteasome assembly. 293T Rpn11-TBHA cells labeled with “heavy (H)” or “light (L)” isotopes were transfected with inactive or WT DYRK2, respectively. Proteasomes were isolated from these cells and analyzed by quantitative mass spectrometry (top). DYRK2-WT strongly phosphorylated Rpt3-T25 (middle), without affecting the relative abundance of each subunit in the purified 26S proteasomes (bottom).(b) In vitro binding of K48-linked tetraubiquitin (K48-Ub4) to the proteasome. Control 293T cells and 293T Rpn11-TBHA cells were subjected to streptavidin pulldown. The purified proteasome was treated with DYRK2-WT or DN in vitro and then incubated with K48-Ub4. Proteasome-bound K48-Ub4 was probed with anti-ubiquitin antibody. Western blot of the ubiquitin receptor Rpn13/ADRM1 shows equal amounts of proteasome in the DYRK2-treated samples.(c) Affinity-purified 26S proteasomes (~ 1 μg) was treated with DYRK2-WT or D275N in vitro. ATPase activity was monitored at 37°C using the malachite green method in the absence or presence of a proteasome substrate, UBL-YFP-ODC (1 μg). **p<0.01, *p<0.05 (two-tailed paired Student's t-test, mean ± s.e.m from n=3 independent experiments).(d) 26S proteasomes from 293T cells overexpressing WT or DN DYRK2 were immobilized on streptavidin beads, washed extensively with reaction buffer containing 1 mM ATP or 1 mM ATPγS, and assayed for Suc-LLVY-AMC cleavage in the same buffers. Reaction was carried out for 10 min at 37°C. **p<0.01 (two-tailed paired Student's t-test, mean ± s.e.m from n=3 independent experiments).Source data for c and d can be found in Supplementary Table 3.

Fig 3: Poly‐GA inclusions sequester the proteasome A, BImmunofluorescence of the proteasome subunit PSMC4 and poly‐GA inclusions in GA175‐GFP‐transfected HeLa cells and GA175‐GFP‐transduced rat primary neurons. To confirm cell viability, the cytoplasm of HeLa cells was stained with HCS CellMask™ Deep Red Stain and neuronal dendrites were labeled with MAP2. Scale bar denotes 20 μm.C, DImmunoblots of HeLa cells that were co‐transfected with HA‐ubiquitin and GFP, GA175‐GFP, GFP‐GR149, PR175‐GFP, GFP‐GP47, and PA175‐GFP and incubated for 48 h and analyzed by densitometry. Scatter plot, mean ± SD. One‐way ANOVA with Tukey's multiple comparisons test. **P < 0.01.E–HFlow cytometry analysis of non‐cell‐autonomous proteasome inhibition using HEK293 UbG76V‐GFP reporter cells co‐cultured for 48 h with GA175‐RFP‐ or RFP‐transfected cells. (E) Two‐color scatter plots, presented as pseudo‐color density plots, with compensated RFP fluorescence plotted on the x‐axis and compensated GFP fluorescence on the y‐axis. A representative experiment out of three independent repeats is shown. (F) Comparisons of the corresponding histograms for compensated RFP and UbG76V‐GFP fluorescence from one representative experiment that shows specific transmission of GA175‐RFP associated with accumulation of UbG76V‐GFP in cells co‐cultured with GA175‐RFP. (G) Accumulation of UbG76V‐GFP signal in non‐transfected receiver cells that were co‐incubated for 48 h with GA175‐RFP‐transfected donor cells compared with RFP control. n = 3 biological replicates. Scatter plot with bar graphs of mean ± SD. Unpaired two‐tailed t‐test with Welch's correction. **P < 0.01. (H) Histograms for UbG76V‐GFP intensity showing separate analysis of RFP‐positive and RFP‐negative receiver cells for the GA175‐RFP condition. RFP gating as indicated in (F). Source data are available online for this figure.

Fig 4: Blockade of Rpt3-T25 phosphorylation impedes cell proliferation(a) Guide RNA design and sequencing verification of Rpt3-T25A knock-in using CRISPR/Cas9. The codon of Thr25 (ACC) is underlined in the WT allele. The single point mutation (A→G) is marked by an arrowhead in the sequencing result, and the resulting Ala25 is highlighted in red.(b) T25A knock-in abolishes T25 phosphorylation. Parental (P) and T25A knock-in clones of MDA-MB-468 cells and HaCaT cells were engineered to stably express Rpn11-TBHA (not shown). After affinity purification, proteasome-associated T25 phosphorylation was probed.(c) Growth curves of the indicated parental and T25A knock-in cells. Results are mean ± s.e.m. from n=3 independent experiments. **p<0.01, *p<0.05, Student's T-test (paired two-tailed test).(d) Stabilization of p27Kip1 and p21Cip1 by T25A knock-in. HaCaT cells were enriched at early S phase with 0.4 mM hydroxyurea (HU) treatment then released into regular medium containing cycloheximide for the indicated lengths of time. Protein levels of p27Kip1 and p21Cip1 were determined from total cell extracts (left). No significant difference in their mRNA levels was seen between the parental and T25A cells at the end of HU treatment. Results are mean ± s.e.m. from n=3 independent experiments (right).(e) Cell cycle analysis of HaCaT cells. After synchronization at G1/S boundary, cells were released and harvested at the indicated time points, stained with propidium iodide and analyzed by FACS. The percentage of cells in G1, S and G2/M phases are shown at the bottom.(f) HaCaT cells were synchronized and released as in (e). Cell lysates collected at each time point were analyzed by western blot.Source data for c and e can be found in Supplementary Table 3.

Fig 5: Rpt3-T25 phosphorylation is required for tumor growth in vivo(a) Tumor xenograft studies with parental and genome-edited MDA-MB-468 cells injected subcutaneously into nude mice. Tumor volumes at each time point after injection are shown (mean ± s.e.m). *p<0.05 (n = 5 mice, One-way ANOVA).(b) Xenograft tumors from (a) were resected at 6 weeks post-injection and imaged. Tumor weights are shown on the right as mean ± SEM. **p<0.01, ***p<0.001 (n =5 mice, compared to parental line, two-tailed non-paired Student's T-test).(c) Histological examination of consecutive sections of the tumors with Ki-67 and hematoxylin/eosin staining. Scale bar = 100 μm.(d) A model of reversible phospho-regulation of the 26S proteasome (adapted from ref.53). The approximate position of Rpt3-T25 in the 26S proteasome complex is highlighted. Cell cycle-dependent Rpt3-T25 phosphorylation regulated by DYRK2 facilitates the degradation of key proteins such as p21 and p27, which in turn promotes cell cycle transition. Pharmacological intervention of this process by targeting proteasome kinases can have therapeutic potentials.