The deubiquitinylation and localization of PTEN are regulated by a HAUSP-PML network

Abstract

Nuclear exclusion of the PTEN (phosphatase and tensin homologue deleted in chromosome 10) tumour suppressor has been associated with cancer progression. However, the mechanisms leading to this aberrant PTEN localization in human cancers are currently unknown. We have previously reported that ubiquitinylation of PTEN at specific lysine residues regulates its nuclear-cytoplasmic partitioning. Here we show that functional promyelocytic leukaemia protein (PML) nuclear bodies co-ordinate PTEN localization by opposing the action of a previously unknown PTEN-deubiquitinylating enzyme, herpesvirus-associated ubiquitin-specific protease (HAUSP, also known as USP7), and that the integrity of this molecular framework is required for PTEN to be able to enter the nucleus. We find that PTEN is aberrantly localized in acute promyelocytic leukaemia, in which PML function is disrupted by the PML-RARalpha fusion oncoprotein. Remarkably, treatment with drugs that trigger PML-RARalpha degradation, such as all-trans retinoic acid or arsenic trioxide, restore nuclear PTEN. We demonstrate that PML opposes the activity of HAUSP towards PTEN through a mechanism involving the adaptor protein DAXX (death domain-associated protein). In support of this paradigm, we show that HAUSP is overexpressed in human prostate cancer and is associated with PTEN nuclear exclusion. Thus, our results delineate a previously unknown PML-DAXX-HAUSP molecular network controlling PTEN deubiquitinylation and trafficking, which is perturbed by oncogenic cues in human cancer, in turn defining a new deubiquitinylation-dependent model for PTEN subcellular compartmentalization.

Figure 1. Aberrant localization of PTEN in APL and Pml-null MEFs

a, Immunofluorescence (IF) analysis of PTEN (green) and PML (red) in AML and APL patient derived bone marrow smears demonstrates cytoplasmic localization of PTEN in APL, but not AML patient samples. Scale bars, 10 μm. Percentage of cases and other representative images of AML and APL are shown in Supplementary Figure 1. b, Immunohistochemical (IHC) analysis of PTEN and PML in APL patient derived biopsies before and after treatment with ATRA shows the restoration of nuclear PTEN localization in vivo by ATRA. Percentage of cases of APL biopsies before and after treatment with ATRA is shown in Supplementary Figure 3a. Inset indicates PML localization (full-size of images are shown in Supplementary Figure 3c). Negative control for PTEN staining is shown in Supplementary Figure 3b. Scale bars, 20 μm. c, Nuclear (N) and cytoplasmic (C) fractionation of PTEN in APL cell line NB4 after treatment with ATO (4 or 24 hours) or ATRA (72 hours). Lamin B and Hsp90 serve as nuclear and cytoplasmic markers, respectively. d, Nuclear-cytoplasmic fractionation of PTEN in ATRA-resistant NB4 (NB4R) cells after treatment with ATO or ATRA. e, IF analysis of Pten and Pml in promyelocytes of wild-type (WT) and MRP8-PML-RARα transgenic (Tg) mice after treatment with ATRA and nuclear-cytoplasmic fractionation analysis of Pten in Tg mice (inset). Scale bars, 10 μm. f, IF analysis of Pten in Pml^+/+ and Pml^-/- MEFs. Scale bars, 10 μm.

Figure 2. HAUSP interacts with and deubiquitinylates PTEN

a, ATRA-induced PTEN monoubiquitinylation. Lysates from HA-tagged KØ-Ub-transfected NB4 cells with either DMSO (lanes 1 and 3) or ATRA (lanes 2 and 4) were immunoprecipitated (IP) with control IgG (lanes 1 and 2) or anti-PTEN (lanes 3 and 4), and then analyzed for monoubiquitinylation by immunoblotting with anti-HA. Molecular weights are in kDa. b, Interaction between PTEN and HAUSP in vivo. Immunobloting of U2OS cell lysates after IP with anti-PTEN or anti-HAUSP antibodies. Asterisks indicate heavy chain of IgG c, Interaction between PTEN and HAUSP in vitro. GST pull-down assay of the purified GST-PTEN protein with ³⁵S-labeled in vitro-translated HAUSP protein. Molecular weights are in kDa. d, Deubiquitinylation of PTEN in vitro by HAUSP. The purified PTEN protein was incubated with the purified recombinant proteins of either wild-type (wt) or mutant form (cs) of HAUSP. Molecular weights are in kDa. e, Increase of PTEN monoubiquitinylation by HAUSP depletion. Lysates from HA-KØ-Ub and either luciferase (control, lanes 1 and 3) or HAUSP siRNA (lanes 2 and 4)-cotransfected U2OS cells were immunoprecipitated (IP) with control IgG (lanes 1 and 2) or anti-PTEN (lanes 3 and 4), and then analysed for monoubiquitinylation by immunoblotting with anti-HA. Molecular weights are in kDa.

Figure 3. HAUSP regulates PTEN localization

a, Regulation of PTEN localization by HAUSP. IF analysis of GFP-PTEN in Myc-HAUSP-transfected PC3 cells (*P<0.01; ^#P<0.05). Arrowhead: HAUSP^high. Asterisk: HAUSP^low. Scale bars, 10 μm. b, Nuclear-cytoplasmic fractionation of GFP-PTEN in empty vector or Myc-HAUSP-transfected PC3 cells. c, Regulation of PTEN localization by HAUSP-mediated deubiquitinylation. IF analysis of GFP-PTEN in HA-KØ-Ub or/and Myc-HAUSP-transfected PC3 cells. Scale bars, 10 μm. d, Nuclear-cytoplasmic fractionation of GFP-PTEN in HA-KØ-Ub or/and Myc-HAUSP-transfected PC3 cells. e, Effects of HAUSP depletion on PTEN localization. IF analysis of GFP-PTEN in control or HAUSP siRNA-transfected PC3 cells (*P<0.01; ^#P<0.05). Scale bars, 10 μm. f, Nuclear-cytoplasmic fractionation of GFP-PTEN in control or HAUSP siRNA-transfected PC3 cells. g, Regulation of PTEN localization by HAUSP loss. IF analysis of PTEN in HAUSP^+/+ and HAUSP^-/- HCT116 cells. Scale bars, 10 μm. h, Nuclear-cytoplasmic fractionation of PTEN in HAUSP^+/+ and HAUSP^-/- HCT116 cells. i, IHC analysis of prostate tumor tissue microarray (TMA) for PTEN and HAUSP (left). Arrows point out nuclear PTEN with low HAUSP and arrowheads indicate cytoplasmic PTEN with high HAUSP. PTEN localization in TMA with specimens from 81 prostate cancer patients reveals positive correlation between cytoplasmic PTEN and HAUSP overexpression (right). Statistical significance is indicated.

Figure 4. PML opposes HAUSP-mediated PTEN deubiquitinylation

a, Inhibition of HAUSP-mediated nuclear exclusion of PTEN by PML. IF analysis of GFP-PTEN in Myc-HAUSP, Flag-PMLIV or PMLIV-RARα-transfected PC3 cells (^#P<0.05). Magnified images (arrows) are shown in Supplementary Figure 12a. b, Inhibition of HAUSP-mediated deubiquitinylation of PTEN by PML. Lysates from immortalized Pml^-/- MEFs transfected with Flag-PTEN alone (lanes 1, 5 and 9), Flag-PTEN and Myc-HAUSP (lanes 2, 6 and 10), Flag-PTEN, Myc-HAUSP and Flag-PMLIV (lanes 3, 7 and 11) or Flag-PTEN, Myc-HAUSP and Flag-PMLIV-RARα (lanes 4, 8 and 12) in the presence of HA-KØ-Ub were immunoprecipitated (IP) with control IgG (lanes 5 to 8) or anti-PTEN (lanes 9 to 12), and then analyzed for monoubiquitinylation by immunoblotting with anti-HA. Molecular weights are in kDa. c, IF analysis of Pten in control or Hausp siRNA-transfected primary Pml^-/- MEFs (*P<0.01; ^#P<0.05). Scale bars, 10 μm. d, Regulation of PTEN localization by DAXX. IF analysis of GFP-PTEN in HA-DAXX-transfected PC3 cells (left). Scale bars, 10 μm. Nuclear-cytoplasmic fractionation of GFP-PTEN in empty vector or HA-DAXX-transfected PC3 cells (right). e, Effects of DAXX depletion on PTEN localization. IF analysis of GFP-PTEN in control or DAXX siRNA-transfected PC3 cells (left). Scale bars, 10 μm. Nuclear-cytoplasmic fractionation of GFP-PTEN in control or DAXX siRNA-transfected PC3 cells (right). f, A model for PTEN monoubiquitinylation and localization by NEDD4-1, HAUSP, DAXX and PML. Cytoplasmic PTEN is monoubiquitinylated (Ub) by NEDD4-1 and subsequently translocated into the nucleus, whereas HAUSP induces deubiquitinylation and nuclear export of PTEN. PML inhibits HAUSP-mediated deubiquitinylation of PTEN and can rescue nuclear PTEN localization through DAXX.