SUMO1 modification of PTEN regulates tumorigenesis by controlling its association with the plasma membrane

Huang, Jian; Yan, Jie; Zhang, Jian; Zhu, Shiguo; Wang, Yanli; Shi, Ting; Zhu, Changhong; Chen, Cheng; Liu, Xin; Cheng, Jinke; Mustelin, Tomas; Feng, Gen-Sheng; Chen, Guoqiang; Yu, Jianxiu

doi:10.1038/ncomms1919

Article
Published: 19 June 2012

SUMO1 modification of PTEN regulates tumorigenesis by controlling its association with the plasma membrane

Jian Huang^1,2^na1,
Jie Yan^1,2^na1,
Jian Zhang³^na1,
Shiguo Zhu¹,
Yanli Wang^1,2,
Ting Shi³,
Changhong Zhu^1,2,
Cheng Chen^1,2,
Xin Liu^1,2,
Jinke Cheng¹,
Tomas Mustelin⁴,
Gen-Sheng Feng⁵,
Guoqiang Chen³ &
…
Jianxiu Yu^1,2,3

Nature Communications volume 3, Article number: 911 (2012) Cite this article

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Abstract

The membrane association of the tumour suppressor phosphatase and tensin homologue (PTEN) is required to oppose the phosphatidylinositol-3-kinase/AKT pathway by dephosphorylation of phosphatidylinositol-3,4,5-triphosphate (PIP3). How cytosolic PTEN interacts with its main substrate, PIP3, localized at the inner face of plasma membrane remains unclear. Here we show that PTEN is covalently modified by SUMO1 at both K²⁶⁶ and K²⁵⁴ sites in the C2 domain of PTEN. SUMO1 modification at K²⁶⁶ located in the CBR3 loop, which has a central role in PTEN membrane association, mainly facilitates cooperative binding of PTEN to the plasma membrane by electrostatic interactions. This results in the downregulation of the phosphatidylinositol-3 kinase/AKT pathway and consequently, suppression of anchorage-independent cell proliferation and tumour growth in vivo. Our data demonstrate a molecular mechanism whereby SUMO1 modification is required for PTEN tumour suppressor function by controlling PTEN membrane association and regulation of the phosphatidylinositol-3 kinase/AKT pathway.

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Introduction

The tumour suppressor gene PTEN (phosphatase and tensin homologue deleted on chromosome 10) is one of the most frequently deleted or mutated in diverse human tumours. PTEN is composed of N-terminal phosphatase domain, C2 domain and C-terminal tail domain (Fig. 1a). PTEN binding to the plasma membrane is a critical regulatory step for PTEN function in antagonizing the phosphatidylinositol-3 kinase (PI3K) signalling pathway by converting phosphatidylinositol 3,4,5-triphosphate (PIP3) to phosphtidylinositol 4,5-biphosphate (PIP2)^1,2. PTEN is mainly found in the cytosol and nucleus. Several studies have shown that under normal conditions, only a small fraction of cytosolic PTEN is dynamically bound to the plasma membrane, because PTEN in a constrained conformation has a low affinity for membrane binding^2,3. It is still poorly understood how cytosolic PTEN interacts with its main substrate PIP3 localized at the inner face of plasma membrane, although several elements/factors, including the CBR3 loop in the C2 domain^4,5,6, N-terminal PIP2-binding motif (Fig. 1a) and PIP2^6,7, C-terminal tail phosphorylations^2,3,8,9,10 and integrity of the phosphatase^2,3, have been suggested to be involved in the membrane binding and activation of PTEN.

**Figure 1: PTEN is SUMOylated at K²⁵⁴ and K²⁶⁶ *in vivo*.**

PTEN activities and functions are regulated by two levels of transcriptional networks^11,12,13 and post-translational modifications, the latter including acetylation, oxidation, S-nitrosylation, phosphorylation, mono- and poly-ubiquitination. Acetylation at K¹²⁵ and K¹²⁸ mediated by PCAF (P300/CBP-associated factor) can block PTEN activity¹⁴. Ubiquitination at K²⁸⁹ and K¹³ is regulated by the E3 ubiquitin ligases NEDD4-1^15,16 and WWP2¹⁷; the polyubiquitinated PTEN protein undergoes rapid degradation, whereas the monoubiquitinated form manifests the increased translocation to the nucleus. Oxidation of disulphide linkage of catalytic C¹²⁴ with C⁷¹ mediated by reactive oxygen species also negatively regulates PTEN catalytic activity¹⁸. S-nitrosylation at the major site C⁸⁷ (minor sites including C⁷¹ and C¹²⁴) by reactive nitrogen species inactivates its lipid phosphatase activity and mediates PTEN degradation via ubiquitination¹⁹. Finally, phosphorylation of PTEN mediated by a series of kinases^{9,20,21,22,23}, such as RhoA-associated kinase on S²²⁹/T²³² and T³¹⁹/T³²¹, glycogen synthase kinase 3β on S³⁶²/T³⁶⁶ and casein kinase 2 on S³⁷⁰/S³⁸⁰/T³⁸²/T³⁸³/S³⁸⁵, can regulate protein stability and function in cells. However, SUMOylation of PTEN has not been reported. Here we identify modification SUMO1, with SUMOylation occurring at two sites K²⁶⁶ and K²⁵⁴ in C2 domain of PTEN. Our data indicate that SUMO1 modification at K²⁶⁶ of PTEN is essential for its tumour-suppressor function.

Results

PTEN can be SUMOylated in cells

We analysed the possible SUMOylation sites of PTEN (Supplementary Fig. S1) and found two consensus motifs IK²⁵⁴VE and LK²⁶⁶KD, the former located in Cβ5 and the latter located in CBR3 loop (²⁶³KMLKKDK²⁶⁹) of the C2 domain of the PTEN protein⁴ (Fig. 1a). It has been reported that endogenous PTEN in MCF7 cells associates with Ubc9²⁴, which is a sole E2-conjugating enzyme essential for SUMOylation. We have confirmed that exogenously tagged PTEN proteins are also associated with Ubc9 in 293T cells (Fig. 1b). Thus, the protein interaction between PTEN and Ubc9 indicates that PTEN could be SUMOylated in cells. To verify this, we took advantage of a HeLa cell line that contains high levels of endogenous PTEN to perform in vivo SUMOylation assays¹¹. As shown in Fig. 1c, affinity pulldown with Ni²⁺-NTA resin greatly enriched SUMOylated PTEN with a size of Mr ~75 kDa (the expected normal size of PTEN is 55 kDa), which is PTEN covalently conjugated with one molecule of SUMO1 (SUMO1–PTEN), in HeLa cells transfected with Flag-Ubc9/His-SUMO1, but not in cells without Flag-Ubc9/His-SUMO1. This result suggests PTEN can be SUMOylated in cells.

Highly SUMOylated endogenous PTEN in SENP1^−/− MEFs

Among the sentrin/SUMO-specific protease (SENP) protein family, SENP1 is the main de-SUMOylation enzyme for SUMO1-conjugated substrates^25,26. We used SENP1^−/− mouse embryonic fibroblasts (MEFs) to examine the above hypothesis. Immunoblotting showed that the levels of PTEN protein were comparable in SENP1^−/− and SENP1^+/+ MEFs. Lysates were also immunoprecipitated with anti-PTEN antibody and then immunoblotted, showing that endogenous SUMO1–PTEN was detected in SENP1^−/− and SENP1^+/+ MEFs with the same size of 75 kDa by both anti-SUMO1 and anti-PTEN antibodies (Fig. 1d). SUMOylation of PTEN is supposed to be more easily observed in SENP1^−/− MEFs than that in SENP1^+/+ MEFs. Indeed, we observed that the SUMO1–PTEN in SENP1^−/− MEFs was four- to six-fold higher than that in SENP1^+/+ MEFs (Fig. 1d). These results demonstrated that endogenous PTEN can be SUMO1-modified cells.

PTEN is modified by SUMO1 at both K²⁶⁶ and K²⁵⁴ sites

To determine the SUMO acceptor sites in PTEN, mutants K²⁵⁴R, K²⁶⁶R or K²⁵⁴R/K²⁶⁶R were generated. We performed SUMOylation assays in 293T cells co-transfected wild-type (WT) PTEN, mutants K²⁵⁴R, K²⁶⁶R or K²⁵⁴R/K²⁶⁶R with or without Flag-Ubc9/His-SUMO1. As expected, one band of SUMOylated Flag-PTEN with a size of Mr ~75 kDa was observed in Ni²⁺-NTA pulldown of cells transfected with PTEN–WT plasmid (Fig. 1e). The double mutant K²⁵⁴R/K²⁶⁶R completely abolished SUMOylation, whereas the single mutants K²⁵⁴R or K²⁶⁶R greatly reduced SUMOylation in comparison with cells transfected with the WT construct, consistent with the notion that both K²⁵⁴ and K²⁶⁶ are bona fide SUMOylation sites. However, we did not observe a shift in PTEN from 55 to 95 kDa, which is presumably conjugated with two molecules of SUMO1—(SUMO1)₂–PTEN (Supplementary Fig. S2), suggesting that K²⁵⁴ and K²⁶⁶ are not simultaneously SUMOylated in cells. Thus, we used only two single (K²⁵⁴R and K²⁶⁶R), but not double, mutants to investigate the physiological function of SUMOylated PTEN. We have confirmed that these two point mutations do not alter other PTEN modifications, such as ubiquitination and acetylation (Supplementary Fig. S3).

PTEN SUMOylation suppresses anchorage-independent growth

To conduct functional analyses, we generated stable PC3^luc cell lines by polyclonal lentiviral infections with Lenti-Vector, PTEN–WT, PTEN–K²⁵⁴R or PTEN–K²⁶⁶R. The expression of PTEN was comparable in all clones when assessed by western blotting (Supplementary Fig. S4a), indicating that these two mutations do not affect PTEN transcription rate and protein stability. Additionally, PTEN proteins, including mutants K²⁵⁴R and K²⁶⁶R, still localized predominantly to the cytoplasm (Supplementary Fig. S4b).

To explore whether PTEN SUMOylation affects the transforming potential of each stable PC3^luc cell lines, we performed a soft agar colony-forming assay in the presence of 10% fetal bovine serum (FBS). As expected, cells transfected with PTEN–WT showed inhibition of colony growth when compared with the Lenti-Vector-transfected cells. PTEN–K²⁶⁶R-transfected cells did not suppress anchorage-independent growth, whereas PTEN–K²⁵⁴R partially lost the ability to suppress growth. Most importantly, the PTEN–K²⁶⁶R-transfected cells produced numbers and sizes of colonies equivalent to those produced by the Lenti-Vector-transfected cells (Fig. 2a,b). We also observed a similar pattern of results with the soft-agar colony-forming assay in the presence of 1% FBS (Supplementary Fig. S5a). Moreover, we generated stable 293T cells expressing PTEN–WT, PTEN–K²⁵⁴R, PTEN–K²⁶⁶R or Lenti-Vector. The colony-forming assays with these stable cell lines showed the similar pattern of results as those in stable PC3 cell lines (Supplementary Fig. S5b).

**Figure 2: PTEN SUMOylation is essential for its suppression of anchorage-independent growth and tumour growth *in vivo*.**

PTEN SUMOylation suppresses tumour growth in vivo

To investigate whether PTEN SUMOylation also influences tumour growth in vivo, each stable PC3^luc cell line was inoculated subcutaneously into the backs of male severe combined immunodeficiency mice. Bioluminescent imaging assessment was performed at day 14 after injection, showing that no tumours were detected in the PTEN–WT group. Tumours in the PTEN–K²⁵⁴R group grew significantly more slowly than those in both the PTEN–K²⁶⁶R and the Lenti-Vector groups, indicating that K²⁵⁴R mutation results in partial loss of the PTEN tumour suppressor activity. Notably, tumours in the PTEN–K²⁶⁶R group grew as fast as those in the Lenti-Vector group, suggesting that K²⁶⁶R mutation leads to the complete loss of PTEN tumour suppressive activity (Fig. 2c,d). These data suggest that K²⁶⁶ SUMOylation is mainly responsible for PTEN function as a tumour suppressor.

SUMO-site mutations impair PTEN downregulation of p-AKT

As PTEN SUMOylation affects tumour growth in both soft agar colonies and in vivo the mouse xenograft model, we attempted to explore whether the mechanism is involved in regulation of AKT phosphorylation. Lysates from stable PC3^luc cells starved for 24 h were immunoblotted, showing that AKT phosphorylation on both T³⁰⁸ and S⁴⁷³ were reduced by 67–69% in PTEN–WT-transfected cells when compared with the Lenti-Vector-transfected cells. In contrast, AKT phosphorylation levels in PTEN–K²⁶⁶R-transfected cells were not significantly different from the Lenti-Vector-infected cells, whereas AKT phosphorylation was reduced by 38–39% in PTEN–K²⁵⁴R-transfected cells when compared with the Lenti-Vector-transfected cells (Fig. 3a,b). The patterns of phospho-AKT were the same as those of findings from both soft-agar colony-forming assay and the in vivo mouse xenograft model described above, demonstrating that K²⁶⁶ SUMOylation of PTEN is required for its capability of tumour suppression through downregulation of phospho-AKT, whereas K²⁵⁴ SUMOylation of PTEN is partially functional. In addition, we have also repeated these experiments in 293T cells stably expressing PTEN–WT, PTEN–K²⁵⁴R, PTEN–K²⁶⁶R or Lenti-Vector (Supplementary Fig. S6), showing the results were very similar to the pattern of results obtained using stable PC3 cell lines.

**Figure 3: PTEN SUMOylation is required for downregulation of AKT phosphorylation.**

A correlation between tumour growth and AKT phosphorylation controlled by PTEN SUMOylation was observed. We stained sections from the mouse xenografts with haematoxylin and eosin, and observed that the morphology of the three xenograft tumours from the Lenti-Vector, PTEN–K²⁵⁴R and PTEN–K²⁶⁶R groups, was not different, whereas no tumour was observed in the PTEN–WT group (Fig. 3c). For these three xenograft tumour sections, we performed immunofluorescence analysis of PTEN and phospho-AKT. As expected, tumours in the Lenti-Vector group had no detectable endogenous PTEN protein, but high phospho-AKT. However, tumours in the PTEN–K²⁶⁶R group displayed high levels of both PTEN and phosphor-AKT, whereas tumours in the PTEN–K²⁵⁴R group showed high PTEN, but moderate phospho-AKT (Fig. 3d). Thus, these data provide evidence that AKT phosphorylation is relevant to tumorigenesis and controlled by PTEN SUMOylation.

Endogenous SUMO1–PTEN effectively reduces phospho-AKT

We asked if highly SUMOylated PTEN can more effectively reduce phospho-AKT in SENP1^−/− MEFs. Indeed, AKT phosphorylation on both T³⁰⁸ and S⁴⁷³ in SENP1^−/− MEFs was approximately seven-fold lower than that in SENP1^+/+ MEFs (Supplementary Fig. S7). Furthermore, to verify whether PTEN is involved in downregulation of phospho-AKT in the SENP1^−/− MEFs system, SENP1^−/− and SENP1^+/+ MEFs stably transduced by Lenti-control-shRNA or Lenti-PTEN-shRNA for PTEN knockdown were serum-starved for 24 h and lysed for the analysis of phospho-AKT. As expected, AKT phosphorylation on both T³⁰⁸ and S⁴⁷³ in control-shRNA-transduced SENP1^−/− MEFs was much lower than those in control-shRNA-transduced SENP1^+/+ MEFs. PTEN was effectively reduced by ~90% by PTEN shRNA in both SENP1^−/− and SENP1^+/+ MEFs. Interestingly, phospho-AKT levels in both SENP1^−/− and SENP1^+/+ MEFs transduced by PTEN shRNA was almost the same and even higher than those in control-shRNA-transduced SENP1^+/+ MEFs (Fig. 3e,f). The MEFs were serum-starved for 24 h, then treated with 10% FBS in a time course from 0 to 60 min for analysis of phospho-AKT. The results showed that phospho-AKT was very low in control-shRNA-transduced SENP1^−/− MEFs (Fig. 3g, the second column), whereas phospho-AKT remained high in PTEN-shRNA-transduced SENP1^+/+ (Fig. 3g, the third column) and SENP1^−/− MEFs (Fig. 3g, the fourth column), in the presence or absence of serum. Phospho-AKT levels were moderate at t=0 and significantly induced by serum in control-shRNA-transduced SENP1^+/+ MEFs (Fig. 3g, the first column). These results fully support the hypothesis that SUMO1–PTEN is required for downregulation of AKT phosphorylation.

SUMOylation facilitates PTEN association with the membrane

To explore the underlying mechanism of SUMOylation at K²⁶⁶ of PTEN that is required for the downregulation of AKT phosphorylation and consequent suppression of tumour growth, we constructed the models of PTEN and SUMO1–PTEN for molecular dynamics (MD) simulations. The simulations were based on the X-ray crystal structures of free PTEN (PDB accession code 1D5R, resolution 2.1 Å)⁴ and SUMO1 (PDB accession code 2IY0, resolution 2.7 Å)²⁵. The structures of PTEN and SUMO1–PTEN (SUMO1 covalently linked to K²⁶⁶ of PTEN) were taken as the starting points for MD simulations. Each MD simulation was carried out using the AMBER suite of programmes (version 8.0) with the parm99 force field (for details, see Methods). On the basis of two equilibrated dynamic trajectories, the snapshots of PTEN and SUMO1–PTEN were respectively extracted and analysed. The results revealed that PTEN in both MD systems are stable and SUMO1 modification is extended from PTEN in the system of SUMO1–PTEN during the simulation time. One eminent feature of charge topology by analysis of the NMR structure of SUMO1 is a stable bipolar surface charge, which on one side displays a positively charged surface and on the other side exhibits a large negatively charged surface²⁷. More recently, it has been reported SUMO1 can bind double-stranded DNA in a sequence-independent manner through electrostatic interaction between SUMO1-positive charge surface and acidic DNA²⁸. It is interesting that SUMO1–PTEN in the short MD simulation show a consensus electropositive interface contributed by K³⁹, K⁴⁵ and K⁴⁶ from SUMO1, and by K²³⁷, K²⁶³ and K³¹³ (Fig. 4a), indicating that SUMO1 modification of PTEN may facilitate cooperative binding of PTEN to the electronegative phospholipid membrane or its substrates by electrostatic interaction.

**Figure 4: SUMO1 modification of PTEN facilitates binding of PTEN to the phospholipid membrane.**

To confirm the above notion that SUMO1 modification of PTEN directly mediates its membrane association and consequently converting PIP3 to PIP2, we conducted a cellular fractionation assay to show that PTEN in the membrane fraction of SENP1^−/− MEFs was four-fold greater than that of SENP1^+/+ MEFs (Fig. 4b,c), indicating that PTEN SUMOylation is required for the recruitment of PTEN to the plasma membrane. We also showed that highly SUMOylated PTEN in SENP1^−/− MEFs intensely associated with the membrane, ablating plasma membrane PIP3 (Fig. 4d; Supplementary Fig. S8). On the contrary, low SUMOylated PTEN in SENP1^+/+ MEFs showed no membrane association, thus leading to PIP3 accumulations of the membrane ruffles. Next, we performed quantitative analysis of 50 cells for each MEFs by ImageJ software, showing that the membrane to cytosol ratios of PTEN proteins were 0.03±0.03 and 0.36±0.09 for SENP1^+/+ and SENP1^−/− MEFs, respectively. Conversely, the membrane to cytosol ratios of PIP3 were 0.26±0.07 and 0.04±0.05 for SENP1^+/+ and SENP1^−/− MEFs, respectively (Fig. 4e). As PIP3 is physiologically the major reaction product of PI3K, with PI(4,5)P2 being the major substrate, we used the PI3K inhibitor LY294002 to treat cells. As shown in Supplementary Fig. S9, the immunofluorescence signal of PIP3 staining was significantly reduced, whereas immunofluorescence signal of PTEN staining was not altered in SENP1^+/+ and SENP1^−/− MEFs. These results support our MD simulation model for PTEN, in which SUMOylation facilitates binding of PTEN to the phospholipid membrane.

K²⁶⁶–SUMO is mainly responsible for PTEN membrane binding

As we have found that PTEN proteins are SUMOylated at K²⁶⁶ and K²⁵⁴, we wanted to know which SUMOylation site is critical for the recruitment of PTEN to the plasma membrane. Thus, we performed the cellular fractionation assays with stable PC3 cell lines. As shown in Fig. 5a, PTEN was highly enriched in the plasma membrane fractions of both PTEN–WT- and PTEN–K²⁵⁴R-transfected cells, although the latter was relatively lower. Surprisingly, PTEN was not recruited to the plasma membrane of PTEN–K²⁶⁶R-transfected cells. We next used a phosphatase assay of diC₈-PIP3 with immunoprecipitated PTEN from the same amount of above plasma membrane fractions of each stable cell line. We show that PTEN–WT-transfected cells had the highest phosphatase activity and PTEN–K²⁵⁴R mutant had an intermediate phosphatase activity, whereas the PTEN–K²⁶⁶R mutant and the Lenti-Vector-transfected cells had a basal level of activity (Fig. 5b). This result also accurately reflected the abundance of PTEN in those fractions. Moreover, immunofluorescence staining also confirmed that PIP3 accumulation at the plasma membrane of PTEN–K²⁶⁶R-transfected cells were also significantly increased to the similar levels as those of the Lenti-Vector-transfected cells. However, PTEN–K²⁵⁴R-transfected cells showed low levels of plasma-membrane-bound PIP3 (Fig. 5c). In addition, we have also repeated the cellular fractionation and phosphatase assays in 293T cells transiently transfected with the empty vector, PTEN–WT, PTEN–K²⁵⁴R or PTEN–K²⁶⁶R plasmids, together with SUMO1 and Ubc9, and showed comparable results (Fig. 5d,e) to those of stable PC3 cell lines. Taken together, the above results correlated well with the tumour suppressor phenotype, including mouse xenograft and soft agar colony, and demonstrated that SUMOylation at K²⁶⁶ is mainly responsible for the recruitment of PTEN to the plasma membrane.

**Figure 5: SUMOylation at K²⁶⁶ but not K²⁵⁴ is responsible for the recruitment of PTEN to the plasma membrane.**

PTEN-controlled tumorigenesis is dependent on K²⁶⁶–SUMO

To investigate the structural basis of the CBR3 loop in PTEN-controlled tumorigenesis, MD simulations, and structural and functional analysis were performed on PTEN and its four mutants. The results show that the conformation of the CBR3 loop could significantly change, owing to sequential multiple replacements of PTEN M-CBR3 (PTEN²⁶³AAGAADA²⁶⁹)⁴. The most greatest deviation of the loop in PTEN²⁶³AAGAADA²⁶⁹ is around residue 266 and the positively charged property of the loop has been eliminated, which could lead to a lower ability to bind the membrane⁴. On the contrary, single mutations at residue 266 had little effect on the perturbation of the CBR3 loop and the whole PTEN conformation (the root-mean-square deviation for the Cα atoms <1.8 Å) in the solvents. These mutations ranged from a similar positively charged residue (PTEN–K²⁶⁶R) to a polar residue (PTEN–K²⁶⁶Q), to a non-polar residue (PTEN–K²⁶⁶A), in comparison with WT PTEN (Fig. 6a). Although the simulation times were limited, the conserved conformations in the PTEN–K²⁶⁶ mutants provide a clue that these various mutants could have the same biological contribution. To verify this, we also generated PC3 cell lines stably expressing PTEN–K²⁶⁶Q, PTEN–K²⁶⁶A or PTEN–G¹²⁹R, and conducted the soft-agar anchorage-independent growth assays with these mutants. Three point mutants K²⁶⁶R, K²⁶⁶Q or K²⁶⁶A produced soft-agar colonies equivalent in size and number to those produced by the Lenti-Vector- and PTEN–G¹²⁹R-transfected cells (as a positive control)²⁹ (Fig. 6b,c). Furthermore, we determined the levels of phospho-AKT, showing that PTEN–WT effectively inhibited AKT phosphorylation on both T³⁰⁸ and S⁴⁷³, whereas all other mutants including K²⁶⁶R, K²⁶⁶Q, K²⁶⁶A and PTEN–G¹²⁹R completely lost this ability (Fig. 6d). Therefore, the tumour formation of PTEN–K²⁶⁶ mutants is ascribed to SUMOylation deficiency instead of a conformational change.

**Figure 6: PTEN-controlled tumorigenesis is K²⁶⁶-SUMO-dependent and not due to changes in PTEN conformation.**

Furthermore, to more strongly support the hypothesis that the mutation of K²⁶⁶ inhibits PTEN SUMOylation-dependent membrane recruitment, but does not alter the conformation of PTEN thereby hindering its recruitment to the plasma membrane, we made Flag-tagged SUMO1–PTEN (WT, K²⁵⁴R or K²⁶⁶R) fusion expression constructs and generated stable PC3 cell lines by lentiviral infections with these new constructs (Fig. 7a). We conducted western blotting analysis of phospho-AKT (Fig. 7b), the soft-agar colony-forming assays (Fig. 7c) and cellular fraction assays (Fig. 7d). Indeed, all these artificially SUMOylated forms of PTEN were equally recruited to the membrane, although relatively less so when compared with PTEN in the cytoplasmic fractions, which is probably due to the fast dynamic association between PTEN and the inner face of plasma membrane. As expected, similar to PTEN–WT–sumo1, both PTEN–K254R–sumo1 and PTEN–K266R–sumo1 reversed the phenotypic observations of inhibition of phospho-AKT (Fig. 7b) and anchorage-independent growth (Fig. 7c). These results demonstrate that PTEN SUMOylation in PTEN-null PC3 cells is indeed important for PTEN membrane recruitment.

**Figure 7: SUMOylated forms of PTEN by gene fusion are recruited to the plasma membrane and reverse the phenotypic observations.**

Discussion

Recent studies have revealed that the presence of SUMOylated proteins occurs not only in the nucleus, but also in other cellular compartments, including the cytoplasm, mitochondria, endoplasmic reticulum and the plasma membrane³⁰. PTEN is mainly found in the cytoplasm as a lipid phosphatase that dephosphorylates its substrate at the plasma membrane, but is also found in the nucleus^15,16. In this study, we have confirmed that PTEN can be modified by SUMO1 in HeLa cells (Fig. 1c), 293T cells (Fig. 1e) and PC3 cells (Supplementary Fig. S2) using His-tagged SUMO1 conjugates bound to Ni²⁺-NTA beads. Importantly, we used immunoprecipitation and western blotting to show the high level of endogenous SUMOylated PTEN in SENP1^−/− MEFs (Fig. 1d).

In fact, PTEN has also been found to directly translocate to the plasma membrane in certain cell lines and under specific conditions^3,20,31. Our finding that SUMO1 modification of PTEN increases PTEN binding to the plasma membrane (Figs 4,5 and 7) may explain why a small fraction of PTEN acts through the dynamic interaction with the inner face of plasma membrane². Most SUMO targets appear to be SUMOylated to a small percentage at steady state; nevertheless, this low-level SUMOylation causes large effects³⁰. Dynamic cycles of SUMOylation and de-SUMOylation transiently occur within seconds³², and PTEN binds to the plasma membrane for a few hundred milliseconds, which is sufficient to dephosphorylate PIP3². Thus, in view of these time frames, it is reasonable to assume that PTEN SUMOylation and PTEN membrane association can occur in this time.

As previous studies have shown^4,5,6, PTEN binds to phospholipid membranes via its C2 domain, in which the CBR3 loop has a central role in the binding. The solvent-exposed loop has four positively charged lysines (K²⁶³, K²⁶⁶, K²⁶⁷ and K²⁶⁹) and two hydrophobic residues (M²⁶⁴ and L²⁶⁵). A mutant of this region, PTEN M-CBR3 (²⁶³AAGAADA²⁶⁹)⁴, shows reduced affinity for membranes in vitro compared with WT PTEN. In this study, we found K²⁶⁶ in the CBR3 loop is SUMO modified, and the K²⁶⁶ mutant has been revealed to contribute to increased tumour formation (Fig. 2c). To predict the structural and functional relationship of K²⁶⁶, the MD simulations on PTEN-WT and its mutants, including K²⁶⁶R, K²⁶⁶A and K²⁶⁶Q, were performed and the results showed that the conformations in the PTEN K²⁶⁶ mutants are conserved in 5-ns trajectories (Fig. 6a). Considering the temporal and spatial features of PTEN function, long simulation of PTEN systems in the presence of a negatively charged bilayer can provide more convincing evidences in the mechanism studies of both CBR3 SUMOylation and membrane recruitment. Nevertheless, a series of very short MD simulations in this study have been employed to simply predict potential effects of PTEN mutants; these need to be verified with further biological experiments. However, these various mutants with the same biological contribution have been validated by the soft agar anchorage-independent growth assays (Fig. 6b,c) and the determination of phospho-AKT levels (Fig. 6d). Therefore, PTEN-controlled tumorigenesis is more likely to be K²⁶⁶–SUMO-dependent, but not due to changes in PTEN conformation.

It has been proposed in the PTEN open/closed model that phosphorylation at S³⁷⁰/S³⁸⁰/T³⁸²/T³⁸³/S³⁸⁵ of the C-terminal tail regulate membrane association^3,33. This model is also involved in binding of the C2 domain to phosphatidylserine⁴ and the PIP2-binding motif to PIP2^7,34. We have provided evidence that a consensus electropositive interface in SUMO1–PTEN facilitates cooperative binding of PTEN with the electronegative phospholipid membrane or its substrates by electrostatic interaction. Phosphorylation leads to a closed conformation that prevents PTEN association with the plasma membrane^9,10; however, if taken into account the electrostatic status, phosphorylation actually supplies negative charges. Therefore, SUMOylation appears to be a positive regulator in controlling PTEN membrane association, whereas phosphorylation is a negative regulator that may neutralize SUMOylation through intramolecular electrostatic interactions. This hypothesis remains to be validated in the future.

We detected enhanced tumour proliferation when K²⁵⁴ was mutated, but this was less than the K²⁶⁶ mutant, which elicits the same tumorigenic phenotype as seen in the PTEN-null condition (Fig. 2). From this observation, it seems that both K²⁶⁶ and K²⁵⁴ are important in regulating tumour formation, but more so when K²⁶⁶ is mutated. According to the principle of electrostatic interaction, the SUMOylation of either K²⁵⁴ or K²⁶⁶ could provide charge to help PTEN in the process of membrane binding. However, K²⁵⁴ is located in the rigid β-strand of the Cβ5 domain and K²⁶⁶ is located in the flexible CBR3 loop; thus, K²⁶⁶ has more potential for conformational changes to induce biological function. This is in agreement with previous studies that the CBR3 loop is necessary for membrane binding. In addition, the SUMO1 protein contains more than 90 residues, and the requirement of space near the modified site is much larger than for other posttranslational modifications (for example, methylation, acetylation, phosphorylation), leading to an unfavourable factor for single SUMOylation of K²⁵⁴ embedded in the Cβ5 domain or dual SUMOylation of adjacent K²⁵⁴ and K²⁶⁶.

In summary, as shown in Fig. 8, our data unravel an unexpected regulatory mechanism that PTEN SUMOylation is required for inhibition of the PTEN/PI3K/AKT pathway. SUMO1 modification of PTEN at K²⁶⁶ mainly facilitates cooperative binding of PTEN to the electronegative phospholipid membrane by electrostatic interaction, then dephosphorylating PIP3 to PIP2, consequently blocking AKT activation and suppressing anchorage-independent cell proliferation and tumour growth in vivo. Our findings might also have important implications in cancer aetiology and cancer therapy, as dysregulation of the PTEN/PI3K/AKT pathway is associated with diverse cancers.

**Figure 8: A novel regulatory mechanism for the PTEN–PI3K–AKT pathway.**

Methods

Cell cultures

Human embryonic kidney 293T, 293FT and HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS; Hyclone), penicillin and streptomycin (Invitrogen) at 37 °C and 5% CO₂. PC3^luc expressing a firefly luciferase can be used for living imaging³⁵. PC3^luc cells were cultured in RPMI1640 in 10% FCS, penicillin and streptomycin at 37 °C and 5% CO₂. MEF cells from SENP1 homozygous null (SENP1^−/−) and WT (SENP1^+/+) mice³⁶, which was provided by Dr J. K. Cheng in SJTU-SM, were cultured in DMEM with 10% FBS. SENP1^+/+ and SENP1^−/− MEFs were generated and maintained according to the protocol of Cheng et al.³⁶ Cell transfection was performed using Lipofectamine 2000 (Invitrogen).

Soft agar colony assay

The effect of PTEN and its mutants on anchorage-independent growth was assessed using a soft agar colony assay. Briefly, this assay was performed in six-well plates with a base of 2 ml of medium containing 10 or 1% FBS with 0.6% Bacto agar (Amresco). Stable PC3^luc transfectants were seeded in 2 ml of medium containing 10 or 1% FBS with 0.35% agar at 1×10³ or 1×10⁴ cells per well and layered onto the base, respectively. The photographs of the cells growing in the plate and of the colonies developed in soft agar were taken, and the number of colonies was scored by ImageJ V1.45 (NIH, USA).

Analysis of SUMO1-modified protein

SUMO1 modification of PTEN was analysed in 293T, HeLa or PC3 cells by the method of His-tagged SUMO1 conjugates binding to Ni²⁺-NTA beads as previously described¹¹. For analysis of endogenous SUMO1–PTEN, SENP1^−/− and SENP1^+/+ MEFs were lysed in NEM-RIPA buffer, and 1500 μg of lysates were used for immunoprecipitation with 5 μl of anti-PTEN (26H9) antibody, then immunoblotted with anti-SUMO1 and anti-PTEN antibodies (dilution 1:1000).

Extraction of membrane/cytosol fractions

A total of 8−10×10⁶ cells were used for each extraction, and the extractions were performed according to the manufacturer's instructions of FractionPREP Cell Fractionation kit (BioVision, CA, USA). Both subcellular fractions were further resuspended in 400 μl of buffer, and usually 10 μl per lane of each cytosolic and membranous protein fractions were loaded on SDS–polyacrylamide gel electrophoresis and western blotted. The anti-IGFIRβ as a membrane marker³⁷ and anti-β-actin as a cytosolic marker³⁸ were used for the loading control.

Phosphatase assays

Equal amounts of membranous fractions from 1×10⁸ of each stable PC3^luc cell line expressing WT PTEN or mutants were used for immunoprecipitation with 5 μl of anti-PTEN (26H9; Fig. 5b). Equal amounts of membranous fractions from 3×10⁷ of each 293T transiently transfected with the empty vector, Flag-PTEN–WT, –K²⁵⁴R or –K²⁶⁶R, together with or without HA-Ubc9 and His-SUMO1, were used for immunoprecipitation with 5 μl of anti-Flag antibody (Fig. 5e). According to the manufacturer's protocol of Malachite Green Phosphatase Assay Kit (Echelon, Salt Lake City, Utah), the phosphatase reactions with water-soluble diC8-PIP3 (Echelon) were performed with the above immunoprecipitated proteins.

Mouse xenograft models

Murine xenografts were established in 6- to 8-week-old male severe combined immunodeficiency mice using PC3^luc cells, which were stably infected with Lenti-Vector, PTEN–WT, PTEN–K²⁵⁴R and PTEN–K²⁶⁶R, respectively. Per injection site, a total volume of 100 μl containing 2.5×10⁶ PC3^luc cells were inoculated subcutaneously into the backs. Tumour volume was assessed by imaging isofluorane-anaesthetized mice with the IVIS system (Xenogen, Alameda, CA). Images were obtained 10 min after intraperitoneal injection of 1.5 mg (approx. 75 mg kg⁻¹) D-luciferin (Xenogen) in 100 μl of PBS. The light emitted by luciferase-expressing tumours was quantified using Living Image Version 2.50 (Wavemetrics, Lake Oswego, OR). A heatmap image corresponding to photons/second/cm²/steradian (blue lowest and red highest) was superimposed over the reflected light image of the animals for tumour localization. A rectangular region of interest encompassing the mouse head and body was replicated between images, images were adjusted to identical luminescent scales, and bioluminescence reported as total flux for the region in photons per second. Animals were imaged on day 14 and 21. The data is represented as the mean±s.e.m. of three independent experiments with five mice each. In vivo bioluminescent numerical data accounting for tumour growth was compared between groups using one-way repeated-measures analysis of variance for significance of intergroup comparisons. Statistical analysis was performed using Prism 5 (GraphPad Software, La Jolla, CA). All animal studies were conducted with the approval and guidance of Shanghai Jiao Tong University Medical Animal Ethics Committees.

Immunofluorescene and confocal microscopy

Immunofluorescene staining method was performed as previously described¹¹. Briefly, SENP1^−/− and SENP1^+/+MEFs were directly seeded into the uncoated 35-mm dishes at a density of 1.0×10⁵ cells. After 24 h, cells were treated with LY294002 (20 μM) or vehicle (DMSO) for 30 min, then washed in PBS once, fixed with 4% freshly prepared formaldehyde in PBS for 8–10 min, and then washed three times with PBS. Cells were permeabilized with 0.1% Saponin (or Triton 100)/PBS for 15 min, blocked in 2.5% normal goat serum in 0.1% Saponin (or Triton 100)/PBS for 30 min–24 h, incubated in the primary antibodies anti-PTEN(138G6, rabbit monoclonal immunoglobulin G, dilution 1:50) and anti-PI(3,4,5)P₃ (Z-P345b, mouse monoclonal immunoglobulin G, dilution 1:50) diluted in blocking solution for 1 h, washed three times with blocking solution and then incubated in the second antibody (Alexa 568 anti rabbit, or Alexa 488 anti mouse, dilution 1:500) in blocking solution for 30 min. The cells were then washed twice with blocking solution, and once with PBS. DAPI (4′,6-diamidino-2-phenylindole) was added for DNA staining.

Images were taken with a Zeiss LSM710 Confocal Microscope (Carl Zeiss, Jena, Germany). All confocal images were analysed and quantified using ImageJ v. 1.45 (http://rsb.info.nih.gov/ij /). The membrane-to-cytosol ratio of respective experiments was calculated on single Z-planes using the methods developed by Janetopoulos et al.³⁹ and Ulrich et al.⁴⁰ For each of MEFs, 50 staining cells were used for quantitative analysis and statistical analysis (Fig. 4d).

Molecular models of PTEN and SUMOylated PTEN

The models of PTEN, SUMOylated PTEN (SUMO1–PTEN) and four mutated PTEN (K²⁶⁶R, K²⁶⁶Q, K²⁶⁶A, M-CBR3--²⁶³AAGAADA²⁶⁹) for the MD simulations were constructed based on the X-ray crystal structures of free PTEN (1D5R, resolution 2.1 Å)⁴ and SUMO1 (2IY0, resolution 2.7 Å)²⁵. The side chains with missing coordinates were reconstructed using the fragment library of the Biopolymer module in Sybyl version 6.8 (Tripos, St Louis, MO). The modified structures were subjected to energy minimization in Sybyl6.8, using the steepest descent method up to the gradient tolerance of 0.05 kcal per mol·Å to relieve possible steric clashes and overlaps of side chains. The three-dimensional structural model of SUMO1–PTEN was built up using the xLeap module in the AMBER suite (version 8.0).

MD simulations

The structures of six models were taken as the starting points for MD simulations. We performed the MD simulation of PTEN–SUMO1 after modelling, to relax the conformation of PTEN–SUMO1 in solution and remove unfavourable contacts arising from the initial rigid-body modelling, whereas other MD simulations on PTEN mutants were used to investigate the structural basis and conformational changes of the CBR3 loop. Each MD simulation was carried out using the AMBER suite of programmes (version 8.0) with the parm99 force field⁴¹. Each structure was prepared by using the xLeap module in AMBER, in which protons were added to the structure. All ionizable side chains were maintained in their standard protonation states at pH 7.0. The proteins were solvated in cubic box of water molecules, with a water thickness extending at least 10 Å apart from the protein surface. To avoid the instability that might occur during the MD simulations, the solvated system was subjected to minimization for 5000 cycles with protein restrained and followed by another 5,000 cycles with the whole system relaxed. Then, the system was gradually heated from 0 to 300 K during the first 60 ps by three intervals, followed by equilibrium for 80 ps under constant volume and temperature condition. Afterwards, the system was switched to constant pressure and temperature condition and equilibrated for 100 ps to adjust the system to a correct density. Finally, the production simulations were carried out in the absence of any restraint under constant pressure and temperature condition and two 5-ns MD simulations were then conducted on the PTEN and SUMO1–PTEN to probe the function of SUMOylation. This protocol was applied to all of the simulation systems.

All the MD simulations were performed using the parallel version of PMEMD in AMBER suit. The particle mesh Ewald method was employed to calculate the long-range electrostatic interactions, whereas the lengths of the bonds involving hydrogen atoms were fixed with the SHAKE algorithm⁴². During the simulations, the integration time step of 2 fs was adopted and structural snapshots were flushed every 500 steps (1 ps). The non-bonded cutoff was set to 10.0 Å, and the non-bonded pair list was updated every 25 steps. Each production simulation was coupled to a 300 K thermal bath at 1.0 atmospheric pressure by applying the Berendsen algorithm. The temperature and pressure coupling constants were set to 2.0 and 1.0 ps, respectively.

Additional information

How to cite this article: Huang, J. et al. SUMO1 modification of PTEN regulates tumorigenesis by controlling its association with the plasma membrane. Nat. Commun. 3:911 doi: 10.1038/ncomms1919 (2012).

Accession codes

Accessions

Protein Data Bank

1D5R
2IY0

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Acknowledgements

This work was supported by grants National Key Program (973) for Basic Research of China (2011CB811303, 2012CB917101, 2009CB918404); National Natural Science Foundation (31071230, 91129726, 81171941); Shanghai Pujiang Program (10PJ1406600); Shanghai Talent Development Fund to Dr. JY(u); and Shanghai Committee of Science and Technology (11DZ2260200). We also acknowledge partial support from State Key Laboratory of Neuroscience, Chinese Academy of Sciences.

Author information

Jian Huang, Jie Yan and Jian Zhang: These authors contributed equally to this work.

Authors and Affiliations

Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory of Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai, 200025, China
Jian Huang, Jie Yan, Shiguo Zhu, Yanli Wang, Changhong Zhu, Cheng Chen, Xin Liu, Jinke Cheng & Jianxiu Yu
Laboratory of Tumor Suppressor Genes and MiRNAs, Institute of Medical Science, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai, 200025, China
Jian Huang, Jie Yan, Yanli Wang, Changhong Zhu, Cheng Chen, Xin Liu & Jianxiu Yu
Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai, 200025, China
Jian Zhang, Ting Shi, Guoqiang Chen & Jianxiu Yu
Infectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, 92037, CA, USA
Tomas Mustelin
Department of Pathology, University of California San Diego, La Jolla, 92093-0864, CA, USA
Gen-Sheng Feng

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Jian Huang
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Contributions

J. H. performed most of the experiments, analysed data. J. Yan generated PTEN lentivirus and infected cells, did some staining of cells and tissues. J. Z. and T. S. built up the three-dimensional structural model of SUMO1–PTEN and provided Figs 4a and 6a. Y. W. and S. Z. performed animal experiments. C. Z., C. C. and X. L. helped with all experiments. J. C. made SENP1^−/− MEFs. G. C., T. M. and G. F. analysed data. J. Yu designed and supervised all aspects of this work and wrote the paper.

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Correspondence to Jianxiu Yu.

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Supplementary information

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Supplementary Figures S1-S9 and Supplementary Methods (PDF 3624 kb)

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Huang, J., Yan, J., Zhang, J. et al. SUMO1 modification of PTEN regulates tumorigenesis by controlling its association with the plasma membrane. Nat Commun 3, 911 (2012). https://doi.org/10.1038/ncomms1919

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Received: 23 January 2012
Accepted: 21 May 2012
Published: 19 June 2012
DOI: https://doi.org/10.1038/ncomms1919

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