WWP2 is an E3 ubiquitin ligase for PTEN - PubMed (original) (raw)

WWP2 is an E3 ubiquitin ligase for PTEN

Subbareddy Maddika et al. Nat Cell Biol. 2011 Jun.

Abstract

PTEN, a lipid phosphatase, is one of the most frequently mutated tumour suppressors in human cancer. Several recent studies have highlighted the importance of ubiquitylation in regulating PTEN tumour-suppressor function, but the enzymatic machinery required for PTEN ubiquitylation is not clear. In this study, by using a tandem affinity-purification approach, we have identified WWP2 (also known as atrophin-1-interacting protein 2, AIP-2) as a PTEN-interacting protein. WWP2 is an E3 ubiquitin ligase that belongs to the NEDD4-like protein family, which is involved in regulating transcription, embryonic stem-cell fate, cellular transport and T-cell activation processes. We show that WWP2 physically interacts with PTEN and mediates its degradation through a ubiquitylation-dependent pathway. Functionally, we show that WWP2 controls cellular apoptosis and is required for tumorigenicity of cells. Collectively, our results reveal a functional E3 ubiquitin ligase for PTEN that plays a vital role in tumour-cell survival.

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Figures

Figure 1

Figure 1

WWP2 interacts with PTEN. (a) Immunoprecipitation (IP) using either control IgG or anti-PTEN antibody was carried out using extracts prepared from 293T cells. The endogenous interaction of WWP2, NEDD4-1 or EDD with PTEN was evaluated by immunoblotting (WB) with their respective antibodies. (b) Immunoprecipitation using control IgG or anti-Flag (PTEN) antibody was carried out using extracts prepared from either parental 293T cells or 293T derivative cells stably expressing Flag-tagged PTEN. The presence of WWP2 or NEDD4-1 in these immunoprecipitates was evaluated by immunoblotting with their respective antibodies. (c) GST pulldown assay was carried out using immobilized control GST or GST-PTEN fusion proteins on agarose beads followed by incubation with extracts prepared from 293T cells. The in vitro interaction of WWP2 with PTEN was assessed by immunoblotting with WWP2-specific antibodies. (d) Schematic representation of N-terminal Flag-tagged full-length PTEN (FL), along with its various deletion mutants (D1–D7). (e) 293T cells were co-transfected with the indicated Flag-tagged PTEN constructs along with those encoding Myc-tagged WWP2, and the interaction between PTEN and WWP2 was determined by immunoprecipitation and immunoblotting with the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S4.

Figure 2

Figure 2

WWP2 regulates PTEN protein stability by polyubiquitylation. (a) Myc-tagged wild-type or a catalytically inactive C838A mutant of WWP2 were expressed in HeLa cells along with Flag–PTEN and HA–ubiquitin (Ub). 24 h post-transfection, cells were treated with MG132 (10 μM) for 6 h and the levels of PTEN ubiquitylation were evaluated by immunoprecipitation of PTEN using anti-Flag antibody followed by anti-HA immunoblotting. (b) A triple-tagged wild-type PTEN and the PTEN tyrosine mutants along with Myc–WWP2 were expressed in 293T cells and the level of PTEN–WWP2 interaction was detected by immunoprecipitation and immunoblotting with the indicated antibodies. The level of PTEN ubiquitylation was determined by immunoblotting with anti-ubiquitin antibodies. (c) HeLa cells were transfected with control siRNA or siRNAs against WWP2, EDD1 and NEDD4-1. Cell lysates prepared after 6 h MG132 (10 μM) treatment were subjected to immunoprecipitaton using anti-PTEN antibodies. The ubiquitylated PTEN was detected with anti-ubiquitin antibody. The protein expression and the specificity of different siRNAs were confirmed by immunoblotting of cell extracts using antibodies as indicated. (d) HeLa cells were transfected with control siRNA or siRNAs against WWP2, EDD1 and NEDD4-1. The protein levels of PTEN were assessed by immunoblotting using anti-PTEN antibody. (e) HeLa cells transiently expressing Flag-tagged PTEN were either transfected with plasmids encoding Myc-tagged WWP2 wild type or C838A mutant. Twenty-four hours post-transfection, cells were treated with cyclohexamide (CHX) and collected at the indicated times afterwards. The protein levels of PTEN were determined by anti-Flag immunoblotting. Uncropped images of blots are shown in Supplementary Fig. S4.

Figure 3

Figure 3

WWP2 activates AKT signalling and regulates stress-induced cell death in a PTEN-dependent manner. (a) DU145 prostate cancer cells were transfected with either control siRNA, WWP2 siRNA or a combination of WWP2 siRNA and PTEN siRNA. 72 h after siRNA transfection, cells were lysed and cell lysates were blotted with the indicated antibodies. Activation of AKT was detected by western blotting with antibody specific to AKT phosphorylated at Ser 473 (anti-pAKT). (b) DU145 prostate cancer cells transfected with the indicated siRNA were either left untreated or treated with doxorubicin. The percentage of apoptotic cells was measured after 36 h of treatment by using Annexin-V staining. The data shown are derived from three independent experiments (±s.d., for doxorubicin treatment P < 0.01 for cells expressing WWP2 siRNA, compared with cells expressing control siRNA or WWP2 and PTEN siRNA; Student’s _t_-test). (c) BPH1 (prostate epithelial-derived cell line) cells were transfected with either wild-type WWP2 or the C838A mutant. The cells were lysed and the activation of AKT was detected by western blotting with antibody specific to AKT phosphorylated at Ser 473 (anti-pAKT). Total AKT and expressed WWP2 proteins were detected by using AKT and Flag antibodies respectively. Actin was used as a loading control. (d) BPH1 cells transiently expressing wild-type or mutant WWP2 were either left untreated or treated with doxorubicin. After 36 h of treatment, apoptotic cells were determined by Annexin-V staining. The data shown are derived from three independent experiments (±s.d., for doxorubicin treatment P < 0.05 for cells expressing WWP2 wild-type, compared with mock treatment; Student’s _t_-test). Uncropped images of blots are shown in Supplementary Fig. S4.

Figure 4

Figure 4

WWP2 is required for tumorigenicity of cells. (a) DU145 cells were stably transfected with either retroviral-based control shRNA or two different WWP2 shRNAs. The expression levels of various proteins were analysed by immunoblotting with their respective antibodies. Actin was used as a loading control. (b) DU145 clones stably expressing control shRNA or WWP2 shRNAs were seeded and analysed for proliferation. The data shown are derived from four independent experiments (±s.d., P < 0.01, compared with cells expressing control shRNA; Student’s _t_-test). (c) DU145 cell lines stably expressing control shRNA or WWP2 shRNA were tested for anchorage-independent growth in a soft-agar colony assay. Viable colonies after 3 weeks were counted and the data (±s.d.) from three independent experiments were presented (P < 0.01, compared with cells expressing control shRNA; Student’s _t_-test). (d) Puromycin-resistant BPH1 prostate epithelial cells stably expressing either WWP2 wild type or the C838A mutant were established and the expression levels of PTEN and WWP2 were detected by the indicated antibodies. (e) A BPH1 parental cell line and BPH1-WWP2 wild-type or C838A mutant cells were analysed for proliferation in a similar way to that described in b. The data shown are derived from four independent experiments (±s.d., P < 0.05, compared with BPH1 parental cell line; Student’s _t_-test). (f) A non-transformed BPH1 cell line along with WWP2 wild-type- or C838A-mutant-expressing BPH1 cells were tested for anchorage-independent growth in a similar way to that described in c, and the data (±s.d.) were presented as summary of three independent experiments (P < 0.05, compared with BPH1 parental cell line; Student’s _t_-test). (g) DU145 stable clones expressing control shRNA or WWP2 shRNA alone or in combination with PTEN siRNA were analysed for proliferation in a similar way to that described in b. The data shown are derived from four independent experiments (±s.d., P < 0.01, compared with cells expressing control shRNA; Student’s _t_-test). (h) DU145 stable cell lines expressing control shRNA or WWP2 shRNA alone or in combination with PTEN siRNA were tested for anchorage-independent growth in a similar way to that described in c and the data (±s.d.) were presented as a summary of three independent experiments (P < 0.05, compared with cells expressing control shRNA; Student’s _t_-test). (i) Control shRNA- or WWP2 shRNA-expressing DU145 stable cells (5×106) were subcutaneously injected into nude mice and the tumour volumes were measured three times per week (±s.d., n = 5, P < 0.05, compared with cells expressing control shRNA; Student’s _t_-test). Uncropped images of blots are shown in Supplementary Fig. S4.

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