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Deubiquitylation and stabilization of PTEN by USP13

Nature Cell Biology volume 15, pages 1486–1494 (2013)Cite this article

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Abstract

The tumour suppressor PTEN is frequently lost in human cancers. In addition to gene mutations and deletions, recent studies have revealed the importance of post-translational modifications, such as ubiquitylation, in the regulation of PTEN stability, activity and localization. However, the deubiquitylase that regulates PTEN polyubiquitylation and protein stability remains unknown. Here we screened a total of 30 deubiquitylating enzymes (DUBs) and identified five DUBs that physically associate with PTEN. One of them, USP13, stabilizes the PTEN protein through direct binding and deubiquitylation of PTEN. Loss of USP13 in breast cancer cells promotes AKT phosphorylation, cell proliferation, anchorage-independent growth, glycolysis and tumour growth through downregulation of PTEN. Conversely, overexpression of USP13 suppresses tumorigenesis and glycolysis in PTEN-positive but not PTEN-null breast cancer cells. Importantly, USP13 protein is downregulated in human breast tumours and correlates with PTEN protein levels. These findings identify USP13 as a tumour-suppressing protein that functions through deubiquitylation and stabilization of PTEN.

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Figure 1: USP13 is a PTEN-interacting deubiquitylase that regulates PTEN and AKT signalling.
Figure 2: USP13 regulates the PTEN protein level but not its subcellular localization.
Figure 3: USP13 directly interacts with and deubiquitylates PTEN.
Figure 4: USP13 stabilizes PTEN protein.
Figure 5: Loss of USP13 promotes tumour growth and glycolysis through downregulation of PTEN.
Figure 6: USP13 suppresses tumorigenesis and glycolysis in PTEN-positive but not PTEN-null breast cancer cells.
Figure 7: USP13 protein is downregulated in human breast cancer and correlates with PTEN protein levels.

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Acknowledgements

We thank J. Yuan, Z. Gong, A. Sorokin, J. Wang, N. Li, L. Feng and L. Li for reagents and technical assistance. This work is supported by US National Institutes of Health grants R00CA138572 (to L.M.) and R01CA166051 (to L.M.) and a Cancer Prevention and Research Institute of Texas Scholar Award R1004 (to L.M.).

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Author notes

  1. Peijing Zhang and Yongkun Wei: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA

    Jinsong Zhang, Peijing Zhang, Hai-long Piao, Wenqi Wang, Min Wang, Dahu Chen, Junjie Chen & Li Ma

  2. Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA

    Yongkun Wei, Yutong Sun & Mien-Chie Hung

  3. Laboratory of Cell Death and Cell Survival, Centre for DNA Fingerprinting and Diagnostics (CDFD), Nampally, Hyderabad 500001, India

    Subbareddy Maddika

  4. Cancer Biology Program, Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, Texas 77030, USA

    Mien-Chie Hung, Junjie Chen & Li Ma

  5. Center for Molecular Medicine and Graduate Institute of Cancer Biology, China Medical University, Taichung 402, Taiwan

    Mien-Chie Hung

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Contributions

J.Z. and L.M. conceived and designed the study and wrote the manuscript. J.Z. performed most of the experiments. P.Z. contributed to DUB library construction and in vitro deubiquitylation assays. Y.W. and M-C.H. performed studies on tissue microarrays of human patient samples. H-l.P. performed xenograft implantation. W.W. and J.C. assisted with tandem affinity purification and mass spectrometric analysis. S.M. provided the PTEN mutant constructs. M.W. assisted with animal care. D.C. assisted with lactate secretion assays. Y.S. maintained shRNA and ORF clones and assisted with glucose uptake assays.

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Correspondence to Li Ma.

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Integrated supplementary information

Supplementary Figure 1 Effects of five PTEN-interacting deubiquitinases on cell proliferation and colony formation.

(a) Five FLAG-tagged DUBs were expressed in MCF7 cells, immunoprecipitated with FLAG beads and immunoblotted with antibodies to PTEN and FLAG. (b) Growth curves of MCF7 cells transduced with USP7, USP8, USP10, USP13 or USP39. (c, d) Images (c) and quantification (d) of anchorage-independent growth of MCF7 cells transduced with USP7, USP8, USP10, USP13 or USP39. Data in (b) and (d) are the mean of 3 wells per group and error bars indicate s.e.m. The experiments were repeated 3 times. Statistical significance was determined by two-tailed, unpaired Student’s t test. Uncropped images of blots are shown in Supplementary Fig. 6.

Supplementary Figure 2 Regulation of PTEN and AKT signaling by USP13.

(a) Immunoblotting of USP13, PTEN and β-actin in a series of human breast cancer cell lines. (b) Immunoblotting of USP13, PTEN, p-AKT, AKT, p-FOXO1/3, FOXO1 and HSP90 in MDA-MB-231 cells transduced with USP13 alone or in combination with PTEN shRNA. (c) Immunoblotting of FLAG–USP13, HA-GFP and β-actin in 293T cells transfected with USP13 shRNA in combination with FLAG-tagged wild-type USP13 or an RNAi-resistant mutant of USP13 (USP13-RE). Co-transfected HA-GFP serves as the control for transfection. (d) Immunoblotting of USP13, PTEN, p-AKT, AKT, p-FOXO1/3, FOXO1 and HSP90 in USP13 shRNA-transduced SUM159 cells with or without ectopic expression of an RNAi-resistant mutant of USP13 (USP13-RE). (e) Immunoblotting of USP13, PTEN, p-AKT, AKT and β-actin in USP13 shRNA-transduced SUM159 cells with or without ectopic expression of PTEN. Cells were serum-starved and treated with 10 ng/ml insulin for 15 minutes. Uncropped images of blots are shown in Supplementary Fig. 6.

Supplementary Figure 3 USP13 does not alter PTEN mRNA levels.

(a) qPCR of PTEN and USP13 in USP13 shRNA-transduced SUM159 cells. (b) qPCR of PTEN in MDA-MB-231 cells transduced with wild-type USP13 or the USP13C345A mutant. Data in (a) and (b) are the mean of 3 triplicates per group and error bars indicate s.e.m. The experiments were repeated 3 times.

Supplementary Figure 4 USP7, USP10, USP8 and USP39 do not regulate PTEN protein levels.

Immunoblotting of the USP, PTEN and HSP90 in SUM159 cells with knockdown of USP7 (a), USP10 (b), USP8 (c) or USP39 (d). Uncropped images of blots are shown in Supplementary Fig. 6.

Supplementary Figure 5 Validation of the PTEN- and USP13-specific antibodies for immunohistochemistry.

The PTEN antibody was validated using BT549 (PTEN-null) and MCF7 (PTEN-positive) cell lines. The USP13 antibody was validated using parental SUM159 cells (USP13-positive) and USP13 shRNA-transduced SUM159 cells (USP13-depleted). To prepare sections, we fixed the cell pellet in formalin and embedded it in paraffin. Brown staining indicates positive immunoreactivity. Scale bar: 50 μm.

Supplementary Table 1 List of PTEN-interacting proteins identified by TAP-MS analysis.
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Supplementary Table 2 List of USP13-interacting proteins identified by TAP-MS analysis.
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Zhang, J., Zhang, P., Wei, Y. et al. Deubiquitylation and stabilization of PTEN by USP13. Nat Cell Biol 15, 1486–1494 (2013). https://doi.org/10.1038/ncb2874

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