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Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis

Nature Cell Biology volume 18, pages 202–212 (2016)Cite this article

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Abstract

Despite the fundamental importance of proteasomal degradation in cells, little is known about whether and how the 26S proteasome itself is regulated in coordination with various physiological processes. Here we show that the proteasome is dynamically phosphorylated during the cell cycle at Thr 25 of the 19S subunit Rpt3. CRISPR/Cas9-mediated genome editing, RNA interference and biochemical studies demonstrate that blocking Rpt3-Thr25 phosphorylation markedly impairs proteasome activity and impedes cell proliferation. Through a kinome-wide screen, we have identified dual-specificity tyrosine-regulated kinase 2 (DYRK2) as the primary kinase that phosphorylates Rpt3-Thr25, leading to enhanced substrate translocation and degradation. Importantly, loss of the single phosphorylation of Rpt3-Thr25 or knockout of DYRK2 significantly inhibits tumour formation by proteasome-addicted human breast cancer cells in mice. These findings define an important mechanism for proteasome regulation and demonstrate the biological significance of proteasome phosphorylation in regulating cell proliferation and tumorigenesis.

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Figure 1: Rpt3-Thr25 is dynamically phosphorylated during the cell cycle.
Figure 2: Blockade of Rpt3-Thr25 phosphorylation impedes cell proliferation.
Figure 3: Loss of Rpt3-Thr25 phosphorylation downregulates 26S proteasome activity.
Figure 4: DYRK2 is the primary Rpt3-Thr25 kinase.
Figure 5: DYRK2 is a positive regulator of proteasome activity.
Figure 6: Mechanisms by which DYRK2 regulates the proteasome.
Figure 7: DYRK2 positively regulates cell growth.
Figure 8: Rpt3-Thr25 phosphorylation is required for tumour growth in vivo.

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Acknowledgements

The authors would like to thank members of the Dixon laboratory and K.-L. Guan, E. Bennett, B. Yang, M. Kaulich and G. Lander for insightful suggestions and discussions of the manuscript. We acknowledge E. Durrant and A. Ly for excellent technical assistance. We thank S. Murata, A. Matouschek, S. Lindquist and W. Becker for providing crucial cDNAs. We owe a debt of gratitude to C. Bashore and A. Martin for the polyubiquitylated GFP substrate. This work was supported by the NIH grants RO1DK018849 to J.E.D., RO1GM074830 to L.H., and 1RO1CA168689 and 1R01CA174869 to J.Y. X.G. was partly supported by the Susan G. Komen postdoctoral fellowship for breast cancer research (KG111280).

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  1. Xing Guo & Zhiping Wang

    Present address: Present addresses: The Life Sciences Institute, Zhejiang University, Hangzhou 310058, China (X.G.); Department of Neurobiology, Zhejiang University School of Medicine, Hangzhou 310058, China (Z.W.).,

Authors and Affiliations

  1. Department of Pharmacology, University of California-San Diego, La Jolla, California 92093, USA

    Xing Guo, Sourav Banerjee, Jing Yang & Jack E. Dixon

  2. Departments of Physiology and Biophysics and of Developmental and Cell Biology, University of California, Irvine, California 92697, USA

    Xiaorong Wang & Lan Huang

  3. Division of Biological Sciences, University of California-San Diego, La Jolla, California 92093, USA

    Zhiping Wang

  4. Departments of Cellular and Molecular Medicine and of Chemistry and Biochemistry, University of California-San Diego, La Jolla, California 92093, USA

    Jack E. Dixon

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Contributions

X.G. and J.E.D. conceived the study. X.G., X.W. and L.H. made the original observation of Rpt3-Thr25 phosphorylation. X.G. designed most experiments. X.G., X.W., Z.W. and S.B. executed the experiments. X.G., X.W., S.B. and L.H. performed data analyses. X.W. and L.H. generated and analysed all mass spectrometry data. Tumour xenograft study was performed by X.G. with J.Y.’s support and guidance. X.G., Z.W. and J.E.D. wrote the paper.

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Integrated supplementary information

Supplementary Figure 3 Phosphorylation of Rpt3 at Thr25.

(a) Endogenous Rpt3-T25 phosphorylation in multiple cell lines. Rpn11-TBHA was stably engineered in each of the cell lines shown. 26S proteasomes were purified from these cells using streptavidin pulldown and treated with or without λ-phosphatase. Phospho-T25 and total Rpt3 were blotted. MEF, mouse embryonic fibroblast. (b) Differential T25 phosphorylation at G1/S and G2/M phases. Four different breast cancer cell lines were treated with either aphidicolin (Aph, 10 μM, 12 hrs) or nocodazole (Ndz, 100 ng/ml, 16 hrs), and pT25 was probed from whole cell extracts. (c) HaCaT cells infected with pLL3.7-HA-Rpt3-WT or T25V (see Supplementary Figure 3) were treated as in (b), and pT25 was probed from whole cell extracts. The complete lack of T25 phosphorylation in the T25V cells indicates efficient replacement of endogenous Rpt3 with the T25V mutant. (d) An estimation of the fraction of Rpt3 that is phosphorylated at T25. HaCaT cells (parental and T25A knock-in, upper panel) and 293A cells (lower panel) were enriched in G2/M phase by sequential treatments with hydroxyurea (0.4 mM, 24 hrs) and nocodazole (100 ng/ml, 8 hrs). Cells were lysed in buffer containing phosphatase inhibitors, and 100 μg of each cell lysate was mixed with 10 μg of anti-pT25 antibody pre-bound to 100 μl of sheep-anti-rabbit IgG-conjugated Dynabeads (Life Technology). T25-phosphorylated Rpt3 was immunoprecipitated for 1 hr at 4 °C. After extensive washes, the immunocaptured phospho-Rpt3 was run side by side with increasing amounts of whole cell lysates (WCL, serving as standards) on SDS-PAGE. Total Rpt3 was blotted using a rabbit polyclonal primary antibody (Bethyl Laboratory) and a mouse-anti-rabbit, light chain-specific secondary antibody (Jackson ImmunoResearch). For HaCaT cells, 5, 10 and 20 μg of WCL were loaded. For 293A cells, 2.5, 5 and 10 μg of WCL were loaded. Intensity of signals were quantified using the ChemiDoc MP imaging system (Bio-Rad) and representative gels are shown. The amount of immunoprecipitated Rpt3 (i.e. T25-phosphorylated) from 100 μg of lysate equals that of total Rpt3 from approximately 15 μg of WCL, in both cell types. Note that the anti-pT25 antibody is very specific for the phosphorylated form and does not pull down Rpt3-T25A (upper panel). Considering that the majority (but not all) of phospho-Rpt3 was immunodepleted from the input sample (data not shown), we conclude that at least 15% of total Rpt3 is phosphorylated at T25 in these cells under this condition.

Supplementary Figure 4 Rpt3-T25A knock-in impedes cell proliferation.

(a) Rpt3-T25A is properly incorporated into the 26S proteasome. MDA-MB-468 parental cells (P) and the two T25A knock-in clones (#4 and #5) were subjected to anti-Rpt3 IP (Bethyl Laboratories). Rpt3, Rpn2 and 20S subunits from the immunoprecipitates and from the whole cell lysates were blotted. (b) Growth curves of the indicated cells stably expressing Rpt3 shRNA and exogenous Rpt3 (WT or T25V). Results are mean ± s.e.m. from n = 3 independent experiments. ∗∗p < 0.01,∗ p < 0.05, two-tailed paired Student’s T-test. Source data can be found in Supplementary Table 3. (c) MDA-MB-468 parental and T25A knock-in cells were synchronized in early S phase by HU and treated with CHX for the indicated periods of time. Cell lysates were probed for p21Cip1 and p27Kip1. (d) MDA-MB-468 parental and T25A knock-in cells were synchronized by aphidicolin and released into regular medium. Cell lysates collected at each time point were used for western blotting. (e) MDA-MB-468 parental and T25A knock-in cells were synchronized by aphidicolin and released into regular medium containing nocodazole. Cell cycle progression was analyzed by FACS using BrdU and PI double labeling. (f) Mild treatment with HU (0.4 mM, 24 hours) or Aph (10 μM, 12 hrs) used in this study does not cause DNA damage response in HaCaT cells. Total cell lysates were probed for γ-H2AX. High-concentration HU treatment (2 mM) was shown as a positive control. (g) T25A knock-in does not cause ubiquitin depletion. HaCaT cells were released from aphidicolin block into S phase. Cells were lysed in SDS buffer with 10 mM N-ethylmaleimide (NEM). Lysates were sonicated and probed for free ubiquitin and ubiquitinated histone H2B.

Supplementary Figure 5 Loss of Rpt-T25 phosphorylation inhibits proteasome activity.

(a) A schematic of the pLL3.7 lentiviral construct used for generating stable lines with simultaneous Rpt3 knockdown and ectopic HA-Rpt3 expression. (b) Replacement of endogenous Rpt3 with HA-Rpt3-T25V reduces proteasome activity in multiple cell lines. Cells transduced with pLL3.7-HA-Rpt3 (WT or T25V) were partly synchronized with nocodazole treatment for 12 hours. After anti-HA IP, 26S proteasome activity was measured using Suc-LLVY-AMC (top), and the immunoprecipitates were then boiled and probed for pT25 and HA-Rpt3 (bottom). Results are mean ± s.e.m. from n = 3 independent experiments. ∗∗p < 0.01,∗p < 0.05, two-tailed paired Student’s T-test. Source data can be found in Supplementary Table 3.

Supplementary Figure 6 DYRK2 as the Rpt3-T25 kinase.

(a) Overexpression of DYRK kinases increases T25 phosphorylation. 293T cells were transfected with the indicated GFP-tagged DYRK kinases. Total cell extracts were probed with the anti-pT25 antibody. GFP alone was used as a negative control. Note that all five DYRK family members could phosphorylate Rpt3-T25 when overexpressed, with DYRK2 and the closely related DYRK3 being the most active against this site. (b) Knockdown of other DYRKs has little effect on T25 phosphorylation. 293T cells stably expressing control or DYRK shRNAs were transfected with HA-Rpt3 (WT). Phospho-T25 was blotted after anti-HA IP. The remaining mRNA levels of each DYRK kinase in the corresponding knockdown cells were determined by qPCR and shown as percentage of that in control cells. (c) DYRK2 expression is regulated by serum starvation and during cell cycle. HaCaT cells were serum-starved (left) or synchronized (right) as described in Fig. 1. DYRK2 protein and mRNA levels were determined by western blot and qPCR, respectively. ∗∗∗p < 0.001,∗p < 0.05 (two-tailed Student’s T-test, mean ± s.e.m. from n = 3 independent experiments). Source data can be found in Supplementary Table 3. (d) Purification of His-DYRK2-WT and D275N (aa. 74-479) shown by Coomassie staining. The inactive D275N mutant lacks co-translational autophosphorylation and therefore migrates faster on gel than the WT kinase. (e) Tandem mass spectrometry identification of Rpt3-T25 phosphorylation (indicated by arrow) from purified 26S proteasomes treated in vitro with DYRK2.

Supplementary Figure 7 DYRK2 positively regulates proteasome activity.

(a) Guide RNA design for DYRK2 targeting and sequencing verification of DYRK2 disruption in MDA-MB-468 cells. Both guide RNAs target regions close to the start codon, and both clones (#1 and #2) have homozygous single nucleotide insertions. The resulting frame shift leads to a premature stop after 83 amino acids in Clone #1 and after 21 amino acids in Clone #2. (b) Proteasome activity (top) and levels of the indicated proteins (bottom) from parental (P) MDA-MB-468 cells and two independent DYRK2 KO clones were determined as in Fig. 3a. ∗∗p < 0.01,∗p < 0.05 (two-tailed Student’s T-test, compared to parental cells, mean ± s.e.m. from n = 3 independent experiments). Source data can be found in Supplementary Table 3. (c) In vitro degradation of casein. DYRK2-treated 26S proteasomes as in Fig. 5f was incubated with BODIPY-labeled casein at 37 °C in the presence of 1 mM ATP. The rate of fluorescence increase is shown as mean ± s.e.m. from n = 3 independent experiments. ∗p < 0.05, two-tailed paired Student’s T-test. Source data can be found in Supplementary Table 3.

Supplementary Figure 8 DYRK2 knockout also sensitizes HaCaT cells to Bortezomib.

(a) Sequencing verification of DYRK2 KO caused by a single nucleotide insertion (highlighted) at the Cas9 cleavage site. (b) Parental and DYRK2 KO HaCaT cells were seeded in triplicates and treated with Bortezomib for 48 hrs, and the MTS assay was performed as in Fig. 7c. ∗∗p < 0.01, Student’s T-test. n.s., non-significant. Source data can be found in Supplementary Table 3.

Supplementary Figure 9 DYRK2 alterations in cancer.

Overview of genetic changes of the DYRK2 gene across a panel of human cancers (http://www.cbioportal.org).

Supplementary Table 1 SILAC/MS of affinity purified 26S proteasome.
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Supplementary Table 2 Sequences of primers, shRNAs and gRNAs.
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Guo, X., Wang, X., Wang, Z. et al. Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis. Nat Cell Biol 18, 202–212 (2016). https://doi.org/10.1038/ncb3289

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