Biochemical Journal

Research article

Crucial role of the C-terminus of PTEN in antagonizing NEDD4-1-mediated PTEN ubiquitination and degradation

Xinjiang Wang, Yuji Shi, Junru Wang, Guochang Huang, Xuejun Jiang

Abstract

PTEN (phosphatase and tensin homologue deleted on chromosome 10), a potent tumour suppressor and multifunctional signalling protein, is under intricate regulation. In the present study, we have investigated the mechanism and regulation of PTEN ubiquitination catalysed by NEDD4-1 (neural-precursor-cell-expressed, developmentally down-regulated 4-1), a ubiquitin ligase for PTEN we identified recently. Using the reconstituted assay and cellular analysis, we demonstrated that NEDD4-1-mediated PTEN ubiquitination depends on its intact HECT (homologous to E6-associated protein C-terminus) domain. Instead of using its WW domains (protein–protein interaction domains containing two conserved tryptophan residues) as a protein interaction module, NEDD4-1 interacts with PTEN through its N-terminal region containing a C2 domain as well as the HECT domain. Strikingly, we found that a C-terminal truncated PTEN fragment binds to NEDD4-1 with higher affinity than the full-length PTEN, suggesting an intrinsic inhibitory effect of the PTEN C-terminus on PTEN–NEDD4-1 interaction. Moreover, the C-terminal truncated PTEN is more sensitive to NEDD4-1-mediated ubiquitination and degradation. Therefore the present study reveals that the C-terminus of PTEN plays a critical role in stabilizing PTEN via antagonizing NEDD4-1-induced PTEN protein decay; conversely, truncation of the PTEN C-terminus results in rapid NEDD4-1-mediated PTEN degradation, a possible mechanism accounting for attenuation of PTEN function by certain PTEN mutations in human cancers.

  • cancer mutation
  • neural-precursor-cell-expressed developmentally down-regulated 4-1 (NEDD4-1)
  • phosphatase and tensin homologue deleted on chromosome 10 (PTEN)
  • protein degradation
  • stability
  • ubiquitination

INTRODUCTION

PTEN (phosphatase and tensin homologue deleted on chromosome 10) was discovered as a tumour suppressor gene that is frequently mutated or deleted in various types of human cancers [1]. Extensive studies have revealed that PTEN plays important roles in many basic cellular processes, and the tumour suppressor activity of PTEN is associated with its functions in regulating cell growth, apoptosis, differentiation, and maintenance of genomic stability [24]. PTEN is also involved in cell migration, cell size control, angiogenesis and chemotaxis [510]. Therefore PTEN is a master regulator of normal cell physiology.

Biochemically, PTEN specifically dephosphorylates PIP3 (phosphatidylinositol 3,4,5-trisphosphate) to PIP2 (phosphatidylinositol 4,5-bisphosphate) [11], thus negatively regulating the PI3K (phosphoinositide 3-kinase)/PKB (protein kinase B; also called Akt) signalling cascade, which is important for cell survival and many other physiological processes. As expected, the lipid phosphatase domain of PTEN is one of the regions clustered for cancer-associated mutations [12]. On the other hand, PTEN may also possess lipid phosphatase-independent antioncogenic and antimetastasis activities [1316], although the molecular basis underlying these functions of PTEN is not clear.

PTEN is a haplo-insufficient tumour suppressor as demonstrated in mouse prostate cancer models [17,18]. In these mouse models, PTEN expression level correlates inversely in an exquisite dose-dependent manner with the prostate cancer progression, its incidence, latency, and the changes in the molecular signature of the PI3K/PKB pathway [18]. Thus the preservation of PTEN protein above certain levels in cells is crucial for suppression of tumour initiation and progression. In human cancers, multiple mechanisms might be involved in loss or decrease of PTEN function in addition to gene mutation and deletion [19]. These mechanisms may include epigenetic silencing via promoter methylation [2022]. Alternatively, post-translational regulation, such as that of PTEN protein stability, can also be a possible mechanism to regulate the biological function of PTEN.

Regulation of PTEN protein stability has been a subject of intensive study in recent years. PTEN is a relatively stable protein, yet its stability has been shown to be down-regulated in certain circumstances such as zinc treatment [23] or by cytoskeleton disruption due to vinculin deficiency [24]. PTEN stability control might be related to its phosphorylation [25,26] and its PDZ domain-binding motif [27,28], both at the C-terminal region of the protein. The relevance of the PTEN C-terminus to its protein stability is also implicated in tumorigenesis. The C-terminal region of PTEN (at the end of exon 8 and the beginning of exon 9) has been found to be frequently mutated in human cancers, resulting in a mutated or truncated PTEN protein [12]. Some of these mutations, however, do not affect the enzymatic activity of PTEN [29]. Therefore why such mutations were selected during cancer development is not clear. Interestingly, because these abnormal PTEN proteins were expressed at a much lower level than wild-type PTEN in transfected cells, it was proposed that these mutants were subjected to accelerated degradation [30,31]. Consistently, expression of some of these PTEN mutants, albeit active biochemically, gave rise to a loss-of-PTEN phenotype in a cell transformation assay exactly as the enzymatically inactive mutants do [30].

The ubiquitin-mediated proteasomal pathway is an important mechanism that regulates protein stability. Recently, we identified the HECT (homologous to E6-associated protein C-terminus)-domain-containing protein NEDD4-1 (neural-precursor-cell-expressed, developmentally down-regulated 4-1) as the first E3 (ubiquitin ligase) for PTEN [32]. We have demonstrated that NEDD4-1 could ubiquitinate PTEN and down-regulate cellular PTEN levels when overexpressed. Importantly, NEDD4-1 could potentiate oncogenic Ras-induced cell transformation in a PTEN-dependent manner, and NEDD4-1 expression levels inversely correlated with PTEN levels in prostate cancer tissues, indicating the proto-oncogenic property of NEDD4-1. In the present study, we reconstituted NEDD4-1-catalysed PTEN ubiquitination de novo by using all recombinant components, and further investigated regulation of PTEN by NEDD4-1 using both cellular experiments and the in vitro-reconstituted assays. We found that the C-terminal tail of PTEN plays a critical role in stabilizing PTEN through its intrinsic inhibitory effect on PTEN–NEDD4-1 interaction and subsequent ubiquitination. Our findings provide new insights into the mechanism why PTEN C-terminal truncating mutation renders the protein unstable in cells, and suggest the potential of NEDD4-1 as a therapeutic target in treating cancers with this type of PTEN mutation.

MATERIALS AND METHODS

Plasmids

hE1 [human E1 (ubiquitin-activating enzyme)] open reading frame was amplified by PCR using IMAGE clone 3543021 (A.T.C.C. no. 10658553) as a template, and was cloned into pFastBacI vector with a 9×His tag for baculoviral expression in insect cells. An E2 (ubiquitin-conjugating enzyme) bacterial expression plasmid, pT7-UbcH5c, was obtained from Dr Moshe Oren (Weizmann Institute of Science, Rehovot, Israel); pCDNA4/TO/PTEN-Myc was from Dr Neal Rosen (Memorial Sloan-Kettering Cancer Center). Other plasmids, pFastBacI-NEDD4-1 with a C-terminal 9×His tag, individual fragments of NEDD4-1 in pET28a, pET28-PTEN-HA, pGEX4T1-PTEN-HA, pGEX4T1-PTEN(1–351), pGEX4T1-PTEN(352–403) and pCDNA4/TO/PTEN(1–351)-Myc were all constructed by PCR cloning; pCDNA3.1-NEDD4-1(C967S) was generated by site-directed mutagenesis from pCDNA3.1-NEDD4-1. All the plasmids were confirmed by DNA sequencing. NEDD4-1 siRNA (small interfering RNA) oligonucleotides (ON-TARGET plus SMART pool) were purchased from Dharmacon.

Transfections

We used the PEI (polyethyleneimine) method for all transfection experiments except for NEDD4-1 siRNA transfection for which we used Lipofectamine™ 2000. PEI transfection was performed as described previously [33].

Recombinant proteins

For recombinant hE1 purification, 1 litre of Sf9 cells at 1.5–2×106 cells/ml was infected with baculovirus expressing hE1 (C-terminal 9×His tag). Cells were collected, 48 h later, by centrifugation at 500 g for 10 min. They were resuspended in 100 ml of low-salt buffer (20 mM Tris/HCl, pH 8.0, 1 mM 2-mercaptoethanol and 50 mM NaCl) containing protease inhibitor cocktails. The cell suspension was incubated in ice for 15 min. The cells were lysed by homogenizing 25 times twice in a Dounce homogenizer with a 10 min interval in ice. After centrifugation at 20000 g for 30 min, 10 mM imidazole was added to the supernatant. Then the supernatant was loaded on to a column with 1 ml of Ni-NTA (Ni2+-nitrilotriacetate) HisBind resin (Qiagen) prewashed with 20 ml of low-salt buffer. The column was washed with 20 ml of low-salt buffer containing 10 mM imidazole, followed by 20 ml of high-salt buffer (20 mM Tris/HCl, pH 8.0, 1 mM 2-mercaptoethanol and 1000 mM NaCl) containing 10 mM imidazole, and 20 ml of low-salt buffer containing 10 mM imidazole again. The recombinant hE1 was eluted with 10 ml of low-salt buffer containing 100 mM imidazole in ten fractions. The protein concentrations were measured and the purity was evaluated by SDS/PAGE. After dialysis against low-salt buffer, the protein was stored as aliquots of 50 μl at −80 °C. UbcH5c was prepared from 4 litres of BL21 (Novagen) transformed with pT7-UbcH5c. The protein expression was induced by 0.2 mM IPTG (isopropyl β-D-thiogalactoside) at 30 °C for 5 h after bacteria grew to a D600 (attenuance at 600 nm) of 0.6–0.8. The recombinant UbcH5c was purified with a 1 ml nickel-affinity column by the same procedure as that described for hE1 except that the bacteria were lysed by sonication in 100 ml of low-salt buffer containing protease inhibitors. HA (haemagglutinin)-tagged rPTEN (recombinant PTEN) was expressed using pET28-PTEN-HA in 4 litres of BL21(DE3) (Stratagene) culture, and expression was induced with 0.4 mM IPTG at 30 °C for 5 h after the culture reached a D600 of 0.6–0.8. The nickel-affinity purification of rPTEN was performed as described for hE1. Then the affinity-purified rPTEN was further purified by FPLC with a 1 ml HiTrap SP column (Amersham Biosciences) as follows. After washing the column with 10 ml of 100 mM NaCl in Buffer A [20 mM Tris/HCl, pH 7.5, 10 mM NaCl and 1 mM DTT (dithiothreitol)], the protein was eluted by a step elution with 400 mM NaCl in Buffer A. After dialysing against Buffer A for 2 h, the rPTEN was supplemented with 10% (v/v) glycerol, 2 mM DTT and 0.5 mg/ml BSA, and stored at −80 °C in small aliquots. GST (glutathione transferase)–PTEN was expressed with pGEX4T1-PTEN-HA in 4 litres of BL21, induced with 0.4 mM IPTG at 30 °C for 4 h after the culture reached a D600 of 0.6–0.8. The bacterial pellet was resuspended in 100 ml of 50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1 mM DTT and 1 mM EDTA supplemented with protease inhibitors. The cells were lysed by sonication. The supernatant after centrifugation at 20000 g for 20 min was loaded on to a 1 ml glutathione–Sepharose 4B column (Amersham Biosciences; catalogue no. 17-0756-01). Then, the column was washed sequentially with 20 ml of 20 mM Tris/HCl (pH 7.5), 1 mM DTT, 1 mM EDTA and 50 mM NaCl (LS7.5E), then 20 ml of 20 mM Tris/HCl (pH 7.5), 1 mM DTT, 1 mM EDTA and 1000 mM NaCl (HS7.5E), and then 20 ml of LS7.5E again. GST–PTEN was eluted with 10 ml of LS7.5E containing 15 mM GSH. The eluent was further purified by FPLC with a 1 ml HiTrap Q column (Amersham Biosciences) as follows. After washing the column with 10–20 ml of Buffer A, the protein was eluted with a salt gradient of 50–500 mM in Buffer A in 15 fractions at 1 ml per fraction. The protein concentrations were measured and the purity was evaluated by SDS/PAGE. After dialysing against Buffer A for 2 h, the protein was supplemented with 10% glycerol, 2 mM DTT and 0.5 mg/ml BSA, and stored at −80 °C in small aliquots. rNEDD4-1 (recombinant NEDD4-1) was prepared from 1 litre of Sf9 cells infected with baculovirus expressing NEDD4-1–9×His with the same procedure as that described for hE1 except for a further purification step by FPLC with a 1 ml HiTrap Q column. The protein was eluted with a 150–400 mM NaCl gradient in Buffer A. After dialysing against Buffer A for 2 h, glycerol was added to a final concentration of 10% (v/v) and DTT was added to a final concentration of 5 mM. The rNEDD4-1 was frozen in liquid nitrogen as 50 μl aliquots and stored at −80 °C. For NEDD4-1 fragments, pET28 constructs expressing the corresponding NEDD4-1 fragments were transformed into Rosetta (DE3) cells (Novagen). The transformant colonies were doubly selected for kanamycin and chloramphenicol resistance. The cell culture was grown from a single colony and always kept under double selection. The induction and purification of His-tagged NEDD4-1 fragments were essentially the same as that described for UbcH5c.

In vitro PTEN ubiquitination

The reaction was carried out at 30 °C for 1 h in a volume of 15 μl containing 40 mM Tris/HCl (pH 7.5), 2 mM DTT, 5 mM MgCl2, 40 μM of wild-type ubiquitin or ko-Ub (lysine-less ubiquitin; Boston Biochem) as indicated, 50 nM hE1, 200–800 nM UbCH5c, 5 mM ATP (Sigma; catalogue no. A-7699), 20 ng of recombinant HA-tagged PTEN, and indicated amounts of purified rNEDD4-1 from baculovirus-infected insect cells. The reaction was then stopped by adding 7 μl of 4×SDS/PAGE sample buffer and boiled for 5 min. The samples were then resolved by SDS/8% PAGE and the ubiquitinated PTEN species were detected by Western blotting against HA tag (monoclonal anti-HA antibody was from Covance, MMS-101R, HA.11).

In vivo ubiquitination assay of PTEN

In vivo PTEN ubiquitination assay was performed under denaturing conditions as described previously for p53 ubiquitination [34] with modifications. Briefly, HEK-293T cells [HEK-293 cells (human embryonic kidney cells) expressing the large T-antigen of SV40 (simian virus 40)] were plated at 2×106 cells per 10 cm plate. PEI transfection was performed the next morning with the following plasmids added in a total of 15 μg of DNA per 10 cm plate as indicated: 2 μg of HA–ubiquitin, 5 μg of Myc-tagged PTEN, with or without 8 μg of pCDNA3.1-NEDD4-1 or pCDNA3.1-NEDD4-1-CS. The cells were harvested in PBS and washed with 10 ml of PBS again. Then, a cytosolic protein fraction was prepared in 200 μl of 20 mM Tris/HCl (pH 7.5), 300 mM NaCl and 1% Triton X-100 supplemented with protease inhibitors (0.5 μg/ml leupeptin, 1 μg/ml pepstatin 2 and 200 μM PMSF) (T7.5Tx-N300-Pi). Then, 40 μl of the lysate was saved for direct Western blot against PTEN. To the 160 μl cytosolic fraction, 16 μl of 10% (w/v) SDS was added to make a final SDS concentration of 1%. The solution was boiled twice for 5 min followed by rigoutous vortex-mixing. After adding 704 μl of T7.5Tx-N300-Pi to reduce the SDS concentration to 0.2%, the lysate was centrifuged at maximal speed for 10 min at room temperature (25 °C). The obtained supernatant was then used for immunoprecipitation with an anti-Myc antibody (9E10) to precipitate the Myc-tagged PTEN protein species. The immunoprecipitates were boiled for 10 min with 30 μl of 2×SDS/PAGE sample buffer. A 25 μl portion of the samples was subjected to Western-blot analysis against HA to detect the HA-tagged ubiquitin conjugated with anti-Myc–PTEN.

GST pull down for mapping the PTEN–NEDD4-1 interaction

The reaction was carried out at 30 °C for 30 min in a volume of 45 μl containing 20 mM Tris/HCl (pH 7.5), 120 mM NaCl, 380 ng of GST or GST–PTEN (full-length PTEN or fragments as indicated in individual reactions) with or without 3 μl of purified NEDD4-1 from HeLa cells. The reaction was then stopped by adding 800 μl of GST-pull-down buffer (20 mM Tris/HCl, pH 8.0, 500 mM NaCl, 1% Triton X-100, 0.02% BSA and 5 mM 2-mercaptoethanol). Then GST–PTEN was pulled down by adding 8 μl of glutathione–Sepharose 4B (Amersham Biosciences) beads into the diluted reaction solution and rotating at room temperature for 40 min. The beads were washed for 5 times by rotating the tubes in 1 ml of GST-pull-down buffer at room temperature, 5 min per wash. The proteins bound to beads were released by boiling in 30 μl of 2×SDS/PAGE sample buffer for 5 min. The presence of NEDD4-1 was detected by immunoblotting against an anti-NEDD4 rabbit polyclonal antibody (BD Biosciences; catalogue no. 550598). For mapping the NEDD4-1 regions that interact with PTEN, 500 ng of GST–PTEN-(1–351) and the T7-tagged NEDD4-1 fragments expressed and purified from Rosetta DE3 cells were used for the GST pull down. The proteins were mixed and incubated at 30 °C for 30 min in the presence of 0.5% Triton X-100, followed by the same procedure of washing steps as that described above. The pulled-down NEDD4-1 fragments were detected by Western blot against an anti-T7 tag antibody.

RESULTS

NEDD4-1-mediated PTEN ubiquitination and degradation depend on the intact HECT domain of NEDD4-1

Our previous study identified NEDD4-1 as the first E3 for PTEN ubiquitination and degradation, and showed that overexpression of NEDD4-1 can accelerate PTEN turnover [32]. To determine whether this is the direct effect of NEDD4-1 E3 activity towards PTEN, we specifically mutated the putative active cysteine residue (Cys-967) to serine based on the sequence alignment of the HECT domains of NEDD4-1 and E6AP (Figure 1A). This mutant form of NEDD4-1, NEDD4-1 CS, should be inactive enzymatically. In the experiment shown in Figure 1(B), co-transfection of PTEN with wild-type NEDD4-1 but not the NEDD4-1 CS mutant in HEK-293T cells caused a down-regulation of PTEN protein levels (lane 2 compared with lane 3; lane 2 compared with lane 4). Examination of PTEN ubiquitination in cells, monitored either by direct PTEN Western blot (Figure 1C, upper panel) or by immunoprecipitation of PTEN followed by Western blot against transfected HA–ubiquitin (Figure 1C, lower panel), indicated that wild-type NEDD4-1 (lane 2) but not the NEDD4-1 CS mutant (lane 3) could promote PTEN ubiquitination in cells. Importantly, treatment of cells with MG-132 (the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-leucinal) further increased PTEN ubiquitination mediated by wild-type NEDD4-1 (compare lanes 4 and 5) but not by the NEDD4-1 CS mutant (compare lanes 4 and 6). Taken together, these results indicate that NEDD4-1 is a bona fide HECT-domain E3 for PTEN, and its enzymatic activity is required for promoting ubiquitin- and proteasome-dependent degradation of PTEN in cells.

Figure 1 Wild-type NEDD4-1 but not its catalytically inactive mutant promotes PTEN degradation and ubiquitination in cells

(A) Sequence alignment of the HECT domain of NEDD4-1 with that of E6AP. The conserved cysteine essential for E3 activity is shaded. (B) Full-length wild-type NEDD4-1 (FN4–HA) but not the catalytically inactive mutant promotes PTEN degradation in cells. HEK-293T cells in 6 cm plates were transfected with 5 μg of either wild-type (wt) or inactive (cs) NEDD4-1-expressing plasmid together with 1 μg of PTEN plasmid and 100 ng of GFP (green fluorescent protein) plasmid. Western-blot analysis was performed with cell lysates obtained 24 h after transfection. GFP serves as a transfection and loading control. (C) Wild-type NEDD4-1 but not the mutant full-length NEDD4-1 promotes PTEN ubiquitination in cells. HEK-293T cells in 10 cm plates were transfected as in (B) with 2 μg of HA–ubiquitin in all the plates and 5 μg of PTEN–Myc DNA and 8 μg of either wt or CS NEDD4-1 DNA respectively. Cells were treated with or without MG-132 at 25 μM for 4 h before harvest. Upper panel: Western-blot analysis with a PTEN monoclonal antibody (Cascade, clone 6H2.1). Lower panel: Western blot for HA–ubiquitin with an anti-HA monoclonal antibody (Covance, MMS-101R, HA.11) after immunoprecipitation with an anti-Myc antibody (9E10). IB, immunoblot: IP, immunoprecipitation.

De novo reconstitution of NEDD4-1-catalysed PTEN ubiquitination in vitro

To investigate the detailed mechanism by which NEDD4-1 ubiquitinates PTEN, we made an effort to fully reconstitute PTEN ubiquitination by NEDD4-1 in vitro. We expressed and purified all the necessary recombinant proteins for an in vitro ubiquitination reaction. As shown in Figure 2(A), we generated highly purified recombinant UbcH5c, C-terminal HA-tagged PTEN (PTEN–HA), hE1 and NEDD4-1. UbcH5c and PTEN–HA were expressed in bacteria, and E1 and NEDD4-1 were expressed using a baculovirus–insect system. With all these proteins, PTEN ubiquitination was reconstituted in vitro. The PTEN ubiquitination was monitored by the appearance of slow-migrating bands of the PTEN–HA substrate after ubiquitin conjugation. Indeed, the rNEDD4-1 can promote PTEN ubiquitination in a dose- and time-dependent manner in vitro (Figure 2B). At as low as 50 nM concentration, a clear NEDD4-1 activity was detected. At this NEDD4-1 concentration, the pattern of PTEN ubiquitin conjugates was predominantly short polyubiquitin chains mixed with multiple monoubiquitinated species [35]. Concentrations of NEDD4-1 higher than 100 nM promoted strong polyubiquitination of PTEN with the ubiquitin conjugates reaching the top of the gel; prolonged reaction time also caused an accumulation of the polyubiquitinated PTEN with 50 nM NEDD4-1 (Figure 2B, compare lanes 3 and 4 with 6 and 7). These results unambiguously demonstrated that NEDD4-1 is an efficient E3 for PTEN. In Figure 2(C), by using a mutated form of ubiquitin that can only support monoubiquitination (ko-Ub), we confirmed that NEDD4-1 can catalyse monoubiquitination of PTEN at multiple lysine residues of PTEN, thus generating low-molecular-mass ladders. On the other hand, since wild-type ubiquitin can support both monoubiquitination and polyubiquitination, the same low-molecular-mass ladder may represent a mixture of multiple-site monoubiquitination and short-chain polyubiquitination, whereas the smear above this ladder can only be caused by polyubiquitination of PTEN (Figure 2C).

Figure 2 Total reconstitution of NEDD4-1-catalysed PTEN ubiquitination in vitro

(A) SDS/PAGE followed by Coomassie Brilliant Blue staining of purified recombinant proteins of hE1, UbcH5c, NEDD4-1 and PTEN–HA. (B) Time- and NEDD4-1-dose-dependent PTEN ubiquitination. In vitro PTEN ubiquitination assay was carried out in the presence of 20 nM hE1, 800 nM UbcH5c-HM and indicated concentrations of NEDD4-1 (rN4) at 30 °C for 60 and 120 min as indicated [T(min)]. (C) Wild-type ubiquitin (wt-Ub) or ko-Ub was used as indicated for measuring NEDD4-1-catalysed PTEN ubiquitination. Whereas ko-Ub can only support monoubiquitination at all possible sites of PTEN, wt-Ub can also support PTEN polyubiquitination. Different time periods (T) and rN4 doses for individual reactions are as indicated. IB, immunoblot.

Mapping the domains required for interaction of NEDD4-1 with PTEN

We have previously shown that full-length PTEN can directly interact with NEDD4-1 [32]. To further define the structural requirement for this interaction, we generated and purified recombinant GST-fusion proteins containing full-length PTEN, the N-terminal region of PTEN spanning the phosphatase and C2 domains (residues 1–351), and the C-terminal regulatory region (residues 352–403) of PTEN (Figures 3A and 3B). Then, a GST pull-down experiment was performed to examine whether they can interact with NEDD4-1 purified from HeLa cell extracts. As shown in Figure 3(C), full-length PTEN could readily pull down NEDD4-1 as we have reported previously [32], whereas the C-terminal fragment of PTEN could not bind to NEDD4-1. Interestingly, the N-terminal fragment of PTEN was not only able to interact with NEDD4-1, but also possessed a stronger binding affinity for NEDD4-1 than the full-length PTEN (compare lane 2 with lane 3). This result indicates that the N-terminal fragment of PTEN contains the NEDD4-1-interacting domain, and it also suggests that the C-terminal region of PTEN may play a negative role in NEDD4-1–PTEN interaction and the consequent NEDD4-1-mediated PTEN ubiquitination.

Figure 3 Mapping of the domain regions of PTEN that can interact with NEDD4-1

(A) Schematic diagram showing GST-fusion proteins containing the full-length (FL), N-terminus (NT, amino acids 1–351) and C-terminus (CT, amino acids 352–403) of PTEN. (B) Inputs of GST-fusion proteins detected by immunoblotting against GST. (C) GST pull down for detecting the interaction of NEDD4-1 with different regions of PTEN. GST pull-down experiment was performed as described in the ‘Materials and methods’ section using native NEDD4-1 purified from HeLa cell extracts. IB, immunoblot.

Conversely, we also mapped the region of NEDD4-1 required for its interaction with PTEN. A series of T7-tagged recombinant proteins containing NEDD4-1 fragments were expressed and purified (Figures 4A and 4B). Because the N-terminal fragment of PTEN (amino acids 1–351) is a stronger binder to NEDD4-1, we performed GST pull-down experiments using GST–PTEN-(1–351) as the bait. As shown in Figure 4(C), both the N-terminal fragment of NEDD4-1 (fragment 1.2, residues 1–250, containing the C2 domain) and the C-terminal HECT-domain-containing fragment (fragment 5, residues 620–1000) can interact with PTEN, and the N-terminal fragment of NEDD4-1 appears to possess a stronger affinity for PTEN. Surprisingly, the WW domains (protein–protein interaction domains containing two conserved tryptophan residues) of NEDD4-1 are not involved in its interaction with PTEN.

Figure 4 Mapping of the domain regions of NEDD4-1 that can interact with PTEN

(A) A diagram showing T7-tagged NEDD4-1 fragments. (B) Inputs of the T7-tagged NEDD4-1 fragments (100 ng) detected by Western blot against T7 tag. Asterisks indicate non-specific products. (C) GST pull down of NEDD4-1 fragments. GST pull down using GST or GST fused with the PTEN N-terminal fragment (amino acids 1–351) (GST–PTEN-NT) was carried out as described in the ‘Materials and methods’ section. The T7-tagged NEDD4-1 fragments pulled down by GST or GST–PTEN-NT were detected by immunoblotting against the T7 tag (IB: T7).

The C-terminally truncated PTEN is more efficiently targeted by NEDD4-1

Given the fact that the N-terminal PTEN fragment binds to NEDD4-1 with higher affinity than the full-length PTEN (Figure 3C), we examined whether the truncated PTEN is also a more efficient substrate for NEDD4-1-mediated ubiquitination. This is indeed the case as shown in both an in vitro-reconstituted assay (Figure 5A) and a cellular ubiquitination assay (Figure 5B). In vitro, when using different doses of NEDD4-1 and reaction times, we found the truncated PTEN was a better substrate than full-length PTEN, because a polyubiquitinated species with a clearly higher molecular mass was generated when the truncated PTEN was used as the substrate, although the truncated PTEN has a lower molecular mass than the full-length PTEN (Figure 5A, compare lane 4 with lane 11 and lane 7 with lane 14). Interestingly, it appears that for shorter time periods, full-length PTEN substrate generated signals for monoubiquitinated or short-chain polyubiquitinated PTEN, whereas truncated PTEN substrate did not generate comparable signals. We suggest that this is because truncated PTEN substrate is more prone to polyubiquitination by NEDD4-1, whereas full-length PTEN is less so, and because the PTEN antibody is more sensitive in detecting low amounts of monoubiquitinated or short-chain polyubiquitinated PTEN, but not as sensitive in detecting long-chain polyubiquitinated PTEN (the antigen is heavily buried when the ubiquitin chain is long). Consistently, in cells, NEDD4-1 overexpression promoted mainly multiple monoubiquitination or short-chain polyubiquitination of full-length PTEN (Figure 5B, lane 7), suggesting a distributive mechanism of the E3 activity of NEDD4-1 on full-length PTEN; in sharp contrast, NEDD4-1 efficiently promoted ubiquitination of the truncated PTEN [PTEN-(1–351)] to yield predominantly polyubiquitinated products without accumulating multiple-monoubiquitinated ones (Figure 5B, lane 11), demonstrating that the truncated PTEN is a much preferred NEDD4-1 substrate than full-length PTEN, and that NEDD4-1 possibly catalyses polyubiquitination of the truncated PTEN with a more processive mechanism. On the other hand, treatment with MG-132 dramatically enhanced polyubiquitination of both truncated PTEN and full-length PTEN (lanes 8 and 12), indicating that both can be targeted at proteasomal degradation by NEDD4-1. As expected, we also directly observed a decrease in expression of the C-terminal truncated PTEN caused by NEDD4-1 overexpression (Figure 5C).

Figure 5 The C-terminal truncated PTEN is more efficiently targeted by NEDD4-1

(A) Ubiquitination of full-length PTEN and the C-terminal truncated PTEN [PTEN-(1–351)] in vitro. In vitro ubiquitination reactions were performed using recombinant GST–PTEN (full-length) or GST–PTEN-(1–351) as the substrates. Concentrations of rNEDD4-1 (rN4) and reaction time were as indicated. (B) Polyubiquitination of PTEN-(1–351) by NEDD4-1 in vivo. HEK-293T cells in 6 cm plates were transfected with 5 μg of vector, full-length PTEN–Myc or PTEN-(1–351)–Myc as indicated with 2 μg of HA–ubiquitin with or without 4 μg of pCDNA3-NEDD4-1-HA. At 20 h after transfection, cells were treated with or without 50 μM MG-132 for 4 h as indicated. Then, the cytosolic fraction of each sample was analysed by Western-blot analysis with the anti-Myc antibody, 9E10. Polyubiquitinated PTENs are indicated, and asterisks represent endogenous c-Myc leaked from nuclei. The lower panel is an experiment similar to that in the upper panel, but with overall weaker ubiquitination. The results of the lower panel further highlighted the difference in ubiquitination patterns of full-length PTEN and truncated PTEN with MG-132 treatment (compare lanes 8 and 12). (C) Down-regulation of the C-terminal truncated PTEN by NEDD4-1 overexpression in cells. PC3 cells in 6 cm plates were transfected with 1 μg of PTEN-(1–351)–Myc (1–351-Myc), 25 ng of pEGFP, with 6 μg of full-length pCDNA3.1-NEDD4-1-HA (FN4–HA) or vector control as indicated. At 24 h after transfection, cells were harvested for Western-blot analysis of PTEN-(1–351) fragment (anti-Myc antibody 9E10) and FN4–HA (anti-HA); the GFP blot served as a loading control. (D) Rescue of the C-terminal truncated PTEN expression by siRNA knockdown of NEDD4-1. HEK-293T cells in 6 cm plates were transfected with 25 ng of pEGFP, 150 ng of full-length PTEN or PTEN-(1–351)–Myc (1–351-Myc) with or without 2 μl of 20 μM siRNA for NEDD4-1 (ON-TARGET SMART pool duplex; Dharmacon. Cytosolic proteins were extracted 24 h later, and 40 μg of total protein was used for Western-blot analysis using the monoclonal anti-Myc antibody 9E10. The transfection efficiency and protein loading were monitored by anti-GFP Western blot. N4i stands for siRNA against NEDD4-1, and Zi for siRNA against LacZ. (E) Proposed model of regulation of NEDD4-1-mediated PTEN ubiquitination. The C-terminus of PTEN (residues 352–403) has an inhibitory effect on interaction of the N-terminus of PTEN (residues 1–351) with NEDD4-1. Therefore loss of this C-terminal region as found in cancer-associated mutations will cause the loss of this inhibitory effect and make the mutated PTEN more sensitive to NEDD4-1-mediated ubiquitination and degradation. Post-translational modifications (such as phosphorylation) on the C-terminal region as well as certain associating proteins might enhance this C-terminal inhibitory effect on NEDD4-1–PTEN interaction. Other PTEN C-terminus-binding proteins might have an opposite effect and thus stimulate PTEN degradation. The labels used are as follows: N, N-terminus; C, C-terminus; Pase, PTEN phosphatase domain; C2n, N-terminal part of the PTEN C2 domain; C2c, C-terminal part of the PTEN C2 domain; C2, NEDD4-1 C2 domain; HECT, NEDD4-1 HECT domain. The four shaded circles stand for the WW domains of NEDD4-1. IB, immunoblot.

Based on these results, we conclude that truncation of the C-terminus of PTEN can result in accelerated PTEN degradation mediated by NEDD4-1. This predicts that knockdown of NEDD4-1 by RNAi (RNA interference) would prevent the C-terminal truncation mutant from degradation in cells. To test this prediction, HeLa cells were transfected with the C-terminally truncated PTEN with or without NEDD4-1 siRNA. As shown in Figure 5(D), the expression level of the truncated PTEN is tremendously lower than that of the full-length PTEN as reported previously [30]. However, co-transfection with NEDD4-1 siRNA significantly restored expression of the truncated PTEN, whereas under the same conditions, there is a modest increase in full-length PTEN expression.

DISCUSSION

In the present study, we investigated the mechanism and regulation of NEDD4-1-catalysed PTEN ubiquitination. First, by using a catalytically inactive NEDD4-1 mutant, we proved that NEDD4-1 is a bona fide HECT-domain E3 for PTEN (Figure 1). This is important because it has been reported that NEDD4-1 can bind to a RING (really interesting new gene)-finger E3, Cbl, and regulates Cbl's stability through ubiquitination [36]. Thus the question arises whether other NEDD4-1-associated E3s (such as Cbl), instead of NEDD4-1 itself, are the direct E3s for PTEN in cells. Our in vivo PTEN ubiquitination analysis using the inactive NEDD4-1 mutant clearly ruled out such a possibility (Figure 1). Our in vitro-reconstituted assay for NEDD4-1-catalysed PTEN ubiquitination corroborated our conclusion from in vivo experiments that NEDD4-1 is sufficient for mediating PTEN ubiquitination on its own (Figure 2).

We found here that NEDD4-1 interacts with PTEN in a unique way involving the C2 and HECT domains of NEDD4-1. NEDD4-1 contains several WW domains, which are the protein interaction modules for the binding of NEDD4-1 with several proteins containing the PPXY motif [37]. Surprisingly, the WW domains of NEDD4-1 are not required for its interaction with PTEN, although there is a PPXY-like motif in the C-terminal region of PTEN. Rather, NEDD4-1 interacts with PTEN via its N-terminal C2 domain-containing region and the HECT domain (Figure 4). This suggests that the C2 domain of NEDD4-1 might play a regulatory role in PTEN ubiquitination. PTEN also possesses a C2 domain. Since C2 domains have been shown to mediate protein interaction in addition to their conventional membrane association function [3840], it is possible that NEDD4-1 and PTEN interact with each other via their corresponding C2 domains. At this point, we are not successful in generating soluble recombinant proteins for these two C2 domains to directly examine this possibility.

Significantly, we found that the C-terminal (residues 352–403) truncated PTEN mutant possesses a higher affinity for NEDD4-1 than the full-length PTEN, and the truncated PTEN is more efficiently targeted for NEDD4-1-mediated ubiquitination and degradation (Figures 3 and 5). These findings might be important because most of the PTEN cancer-associated mutations are truncation mutations (72%) and a fraction of these truncating mutants lack a partial C2 domain and the entire C-terminus [12]. Our results suggest that the C-terminus of PTEN possesses an intrinsic inhibitory effect on interaction of PTEN, through its C2 domain-containing N-terminus, with NEDD4-1. This finding is consistent with a recent report showing that a PTEN C-terminal fragment (residues 335–403) can actually interact intramolecularly with a short region (residues 255–280) of its N-terminal C2 domain (residues 186–351) [41], which is close to a ubiquitination site of NEDD4-1 in the C2 loop (Lys-289) [35]. Thus this intramolecular interaction between the C-terminus and the C2 domain of PTEN would mask the potential interaction site for NEDD4-1. On the basis of this information, we propose the following model for regulation of PTEN stability by NEDD4-1 (Figure 5E): for intact PTEN protein, its C-terminal region is engaged in an intramolecular interaction with its C2 domain that prevents NEDD4-1-mediated PTEN degradation and results in a long half-life of PTEN, even in the presence of NEDD4-1; the regulatory events on the PTEN C-terminus such as post-translational modification or binding of other factors will alter the accessibility of NEDD4-1 to the PTEN molecule, thereby modulating PTEN stability. This model is consistent with the previous findings that the C-terminus of PTEN and its phosphorylation are important for PTEN stability [25,26,28], and that multiple PTEN C-terminal binding proteins affect PTEN stability. For example, binding of MAGI-2 (membrane-associated guanylate kinase with inverted domain structure-2) has been shown to stabilize PTEN [26], and binding of PICT1 (protein interacting with C-terminal tail 1) to the PTEN C-terminus has a similar effect [42,43]. Furthermore, there are many cancer-associated C-terminal truncation mutants of PTEN. Although most of them have defective C2 domains and are thus inactive, a small percentage of them do have intact C2 domains and are enzymatically active [29]. Our model provides a mechanistic explanation as to why they are selected in human cancers, and why these mutants are difficult to express in cell cultures [12,30]: because such a truncation exposes the N-terminal region of PTEN to a more efficient NEDD4-1 binding and subsequent rapid ubiquitination and degradation of the tumour suppressor. More speculatively, even for the cancer-associated truncation mutants with a defective C2 domain, it is possible that they possess certain enzymatic activity-independent tumour-suppressive functions; thus the rapid degradation mediated by NEDD4-1 can cause a more complete loss of their tumour-suppressive function in human cancer. However, more work is needed to define the enzymatic activity-independent PTEN function and its biological significance.

Our findings on the biochemical basis of NEDD4-1 PTEN functional interaction may have potential therapeutic implications, considering that loss of PTEN expression is a more common phenotype than PTEN gene deletion/mutation in certain types of human cancer [44]. Ideally, this NEDD4-1-targeting therapy can be best tested using human cancer cell lines bearing the wild-type PTEN gene but with low PTEN protein levels. Unfortunately, the previous relevant study dealt mainly with clinical cancer samples instead of cancer cell lines. Nevertheless, our NEDD4-1 RNAi knockdown results suggest that targeting NEDD4-1 for rescuing PTEN expression in cancer deserves further investigation.

Acknowledgments

This work was supported by an American Cancer Society Scholar grant (RSG-07-081-01; to X. J.) and a Geoffrey Beene Cancer Research grant (to X. J.).

Abbreviations: DTT, dithiothreitol; E1, ubiquitin-activating enzyme; E3, ubiquitin ligase; GFP, green fluorescent protein; GST, glutathione transferase; HA, haemagglutinin; hE1, human E1; HECT, homologous to E6-associated protein C-terminus; HEK-293 cells, human embryonic kidney cells; HEK-293T cells, HEK-293 cells expressing the large T-antigen of SV40 (simian virus 40); IPTG, isopropyl β-D-thiogalactoside; ko-Ub, lysine-less ubiquitin; NEDD4-1, neural-precursor-cell-expressed developmentally down-regulated 4-1; PEI, polyethyleneimine; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B (also called Akt); PTEN, phosphatase and tensin homologue deleted on chromosome 10; rNEDD4-1, recombinant NEDD4-1; RNAi, RNA interference; rPTEN, recombinant PTEN; siRNA, small interfering RNA; WW domain, protein–protein interaction domain containing two conserved tryptophan residues

References

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