ATM-mediated PTEN phosphorylation promotes PTEN nuclear translocation and autophagy in response to DNA-damaging agents in cancer cells
Jing-Hong Chen,1,2,† Peng Zhang,4,† Wen-Dan Chen,1,† Dan-Dan Li,1 Xiao-Qi Wu,5 Rong Deng,1 Lin Jiao,1
State Key Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine; Cancer Center;
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1.
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Xuan Li,1 Jiao Ji,1 Gong-Kan Feng,1 Yi-Xin Zeng,1 Jian-Wei Jiang,3* and Xiao-Feng Zhu 1*
Sun Yat-sen University; Guangzhou, China
Department of Hematology; The Second Affiliated Hospital; Guangzhou Medical University; Guangzhou, China
3
Department of Biochemistry; The School of Medicine; Jinan University; Guangzhou, China
4
Department of Microbiology and Immunology; The School of Medicine; Jinan University; Guangzhou, China
5
Department of Science and Research; The Third Affiliated Hospital; Sun Yat-sen University; Guangzhou, China
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2
† These authors contributed equally to this work
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*Correspondence to: Xiao-Feng Zhu; Email: [email protected]; Jian-Wei Jiang; Email:[email protected]
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Keywords: ATM, autophagy, DNA damage, PTEN, topotecan Abbreviations: AKT/PKB, v-akt murine thymoma viral oncogene homolog; AMPK, protein kinase,
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AMP-activated; ATG, autophagy-related; ATM, ATM serine/threonine kinase; Baf.A1, bafilomycin A1;
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CASP3, caspase 3, apoptosis-related cysteine peptidase; CCND1, cyclin D1; CDDP, cisplatin; CENPC/CENP-C, centromere protein C; CITED1/p300/CBP, Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 1; CSNK2/CK2, casein kinase 2; DSBs, DNA double-strand breaks; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GLTSCR2/PICT-1, glioma tumor suppressor candidate region gene 2; GSK3B, glycogen synthase kinase 3 beta; GST, glutathione S-transferase; H2AFX, H2A histone family, member X; JUN, jun proto-oncogene; 1
MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MTORC1, mechanistic target of rapamycin complex 1; MVP, major vault protein; NC, normal control; NEDD4, neural precursor cell expressed, developmentally down-regulated 4, E3 ubiquitin protein ligase; PAGE, polyacrylamide gel electrophoresis; PARP, poly (ADP-ribose) polymerase 1; PI3K, phosphoinositide 3-kinase; PMSF, phenylmethanesulfonyl fluoride; PPase, protein phosphatase; PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5)-trisphosphate; PTEN,
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phosphatase and tensin homolog; RAD51, RAD51 recombinase; RPS6KB/p70S6K; ribosomal protein S6
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kinase, 70kDa; SDS, sodium dodecyl sulfate; SESN2, sestrin 2; siRNA, small interfering RNA; SQSTM1/p62, sequestosome 1; TP53, tumor protein p53; TPT, topotecan; TUBA4A, tubulin, alpha 4a; WT,
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wild type; YFP, yellow fluorescent protein
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PTEN (phosphatase and tensin homolog), a tumor suppressor frequently mutated in human cancer, has various cytoplasmic and nuclear functions. PTEN translocates to the nucleus from the cytoplasm in response
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to oxidative stress. However, the mechanism and function of the translocation are not completely understood. In this study, topotecan (TPT), a topoisomerase I inhibitor, and cisplatin（CDDP）were employed to induce
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DNA damage. The results indicate that TPT or CDDP activates ATM (ATM serine/threonine kinase), which
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phosphorylates PTEN at serine 113 and further regulates PTEN nuclear translocation in A549 and HeLa cells. After nuclear translocation, PTEN induces autophagy, in association with the activation of the
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p-JUN-SESN2/AMPK pathway, in response to TPT. These results identify PTEN phosphorylation by ATM
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as essential for PTEN nuclear translocation and the subsequent induction of autophagy in response to DNA damage.
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Introduction PTEN/MMAC1 (phosphatase and tensin homolog) has been identified as a tumor suppressor gene from a variety of cancers that display frequent deletion of its location on human chromosome 10q23, a locus that is highly susceptible to mutation in primary human cancers and cancer cell lines.1 PTEN phosphatidylinositol
(3,4,5)-trisphosphate
(PtdInsP3)
to
phosphatidylinositol
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converts
(4,5)-bisphosphate in the cytoplasm, thereby directly antagonizing the activity of phosphoinositide
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3-kinase (PI3K).2,3 PTEN inactivation results in the constitutive activation of the PI3K-AKT pathway
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and a subsequent increase in protein synthesis, cell cycle progression, migration, and survival.4,5 PTEN is localized to the nucleus and cytoplasm. Early immunohistochemical studies of PTEN
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localization found that the endogenous wild-type PTEN protein is exclusively localized in the cytoplasm of various cancer cell lines.6 Subsequently, multiple reports have identified PTEN nuclear or
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perinuclear PTEN in normal primary neurons and endothelial cells, PTEN-positive primary breast
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carcinoma samples, normal epithelial and follicular thyroid cells, follicular thyroid adenomas, and thyroid carcinomas.7,8 However, the nuclear PTEN immunostaining is relatively weak in thyroid
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carcinomas compared with normal thyroid follicular cells and follicular thyroid adenomas.9
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Although normal islet cells exhibit predominantly nuclear PTEN immunostaining in pancreatic tissue, 19 of 23 sporadic endocrine pancreatic tumors predominantly display cytoplasmic PTEN expression patterns.10
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Whiteman observes a similar phenomenon, finding increased nuclear PTEN expression in normal melanocytes and reduced nuclear PTEN expression in melanoma cells. However, PTEN protein expression is primarily localized to the cytoplasm of metastatic melanoma samples. An association between the lack of nuclear PTEN and mitotic index has been reported; nuclear PTEN is evident in quiescent cells, but PTEN primarily localizes to the cytosol of actively dividing cells.11 Generally, PTEN is primarily localized to the nucleus in normal quiescent tissues, whereas cytoplasmic PTEN is predominately found in neoplastic 3
tissues. Thus, nuclear PTEN appears to be essential for tumor suppression. Despite the absence of a classic nuclear localization signal, PTEN enters the nucleus by several mechanisms, including simple diffusion, export dependent on a putative cytoplasmic localization signal, and active shuttling by the RAN GTPase or MVP (major vault protein).12-15 PTEN monoubiquitination at Lys13 or Lys289 by NEDD4 increases its nuclear localization, whereas polyubiquitination by NEDD4 promotes its
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cytoplasmic degradation.16,17
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Accumulating evidence suggests multiple mechanisms of PTEN nuclear import depending on the cell type and cellular environment. Moreover, the role of nuclear PTEN is not the same as that of cytoplasmic
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PTEN. Nuclear PTEN plays a crucial role in regulating cellular homeostasis and stability by downregulating
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the MAPK signal pathway, reducing CCND1 (cyclin D1) levels and G0-G1 arrest, upregulating RAD51 (RAD51 recombinase) levels and double-strand-break repair, and interacting with CENPC/CENP-C
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(centromere protein C), which specifically enhances centromere stability and overall genomic stability by promoting CITED1/p300/CBP(Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal
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domain, 1)-mediated acetylation of TP53/p53 (TRP53 in mice) in response to DNA damage and
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apoptosis.18-23
In this study, we find that ATM (ATM serine/threonine kinase) phosphorylates PTEN at serine 113,
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further promoting PTEN nuclear translocation. PTEN nuclear translocation induces autophagy through
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activation of the p-JUN-SESN2-AMPK pathway following treatment with the DNA-damaging agent topotecan (TPT) in A549 and HeLa cells.
Results
DNA-damaging agents induced ATM phosphorylation 4
TPT, which causes DNA damage, is commonly used for cancer treatment in the clinic. CDDP (cisplatin) is also an anticancer drug that causes cross-linking of deoxyribonucleic acid. ATM kinase is activated in response to DNA double-strand breaks (DSBs). To investigate whether TPT or CDDP treatment induces ATM phosphorylation in A549 and HeLa cells, we determined the levels of phosphorylated ATM by immunoblotting. As shown in Fig. S1, ATM phosphorylation increased in a dose- and time-dependent
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manner after treatment with various concentrations of TPT or CDDP for 24 h and with 3 mg/mL TPT or 6
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phosphorylation and activation in A549 and HeLa cells.
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mg/mL CDDP for various times, respectively. These data indicated that TPT and CDDP induced ATM
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ATM regulates PTEN nuclear translocation, following exposure to DNA-damaging agents It has been previously reported that PTEN accumulates in the nucleus of cells treated with apoptotic
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stimuli, including etoposide, doxorubicin,20 or H2O2, indicating that oxidative stress promotes PTEN nuclear accumulation.24 PTEN is normally found in the cytoplasm in A549 and HeLa cells. After A549 and HeLa
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cells were treated with 3 mg/mL TPT for 24 h, endogenous PTEN translocated from the cytoplasm to the
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nucleus. However, when A549 and HeLa cells were treated with 3 mg/mL TPT and 10 mM KU55933 (ATM specific inhibitor) for 24 h, PTEN nuclear translocation was clearly suppressed (Fig. 1A).
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To detect exogenous PTEN nuclear translocation, the 3×Flag-PTEN-WT plasmid was constructed. The
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3×Flag-PTEN-WT expression vector was transiently transfected into HeLa cells, and the cells were treated with TPT (3 μg/mL) and/or KU55933 (10 μM) for 24 h. The transfection and expression efficiency were determined by immunoblotting (Fig. 1B). When HeLa cells were treated with 3 mg/mL TPT for 24 h, exogenous PTEN translocated from the cytoplasm to the nucleus, consistent with the endogenous PTEN translocation pattern observed by confocal microscopy under the same conditions. When the ATM specific inhibitor KU55933 or transient transfection with ATM siRNA was used to inhibit ATM activity, the PTEN 5
translocation was clearly suppressed (Fig. 1C). Also, when HeLa cells were treated with 6 mg/mL CDDP for 24 h, exogenous PTEN translocated from the cytoplasm to the nucleus, which was consistent with the result from TPT treatment (Fig. 1D). To further confirm PTEN nuclear translocation, nuclear and cytoplasm proteins of A549 cells and HeLa cells were extracted, and the PTEN expression in these components was detected by western blotting.
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As expected, PTEN levels were increased in the nuclear fraction compared with the cytosolic fraction in the
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TPT-treated group, suggesting that TPT induces PTEN nuclear translocation. However, TPT-induced PTEN nuclear translocation was significantly inhibited upon treatment with KU55933 and ATM siRNA (Fig. 1E
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and F), indicating that PTEN nuclear translocation is regulated by ATM.
ATM regulates PTEN phosphorylation, following exposure to DNA-damaging agents
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Phosphorylation is an important protein posttranslational modification. Given that we demonstrated that TPT induces ATM phosphorylation and that ATM further regulates TPT-induced PTEN nuclear
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translocation in A549 and HeLa cells, we wanted to determine whether PTEN nuclear translocation was
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regulated by ATM via phosphorylation.
To determine whether ATM directly phosphorylates PTEN, we first analyzed the PTEN sequence using
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Scansite software (http://scansite.mit.edu) and identified a potential ATM phosphorylation site at serine 113
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(Ser113) (Fig. 2A). Next, we developed a phospho-specific antibody against PTEN (Ser113). To validate the Phospho-PTEN (Ser113) antibody, A549 cells were treated with: 1) 3 mg/mL TPT only; 2) 3 mg/mL TPT and l-phosphatase; 3) 3 mg/mL TPT, l-phosphatase, and protein phosphatase (PPase) inhibitors. The phospho-PTEN (Ser113) levels in A549 cells treated with l-phosphatase were significantly decreased compared with the TPT group. The inhibitory effect was partly restored when PPase inhibitors were applied (Fig. 2B). The results confirmed the specificity of the phospho-PTEN (Ser113) antibody. 6
To determine whether TPT induces PTEN phosphorylation at Ser113, we transiently transfected HeLa cells with the 3×Flag-PTEN-WT plasmid. The cells were then exposed to different concentrations of TPT or CDDP for 24 h. The results indicated that the levels of p-PTEN (Ser113) were enhanced in a concentration-dependent manner (Fig. 2C). In addition, A549 or HeLa cells were treated with 3 μg/mL TPT for 24 h, and immunoprecipitation was
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performed using a Flag antibody. The precipitates were subjected to SDS-PAGE and probed with the
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p-PTEN (Ser113) antibody. As shown in Fig. 2D, the p-PTEN (Ser113) levels in the TPT treatment group were significantly increased compared with the control group, indicating that TPT induces PTEN (Ser113)
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phosphorylation.
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Furthermore, we sought to investigate whether TPT-induced PTEN phosphorylation was regulated by ATM. A549 cells and HeLa cells, with reduced ATM expression by siRNA or KU55933, were treated with
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3 mg/mL TPT for 24 h. As shown in Fig. 2E, the p-ATM and p-PTEN (Ser113) levels were significantly increased in the TPT treatment group. However, knockdown of ATM by siRNA or KU55933 significantly
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reduced the p-PTEN (Ser113) levels. These data indicate that ATM regulates TPT-induced PTEN (Ser113)
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phosphorylation.
The in vitro kinase assays with purified proteins was used to assess whether PTEN was directly
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phosphorylated by ATM. As show in Fig. 2F, the p-PTEN (Ser113) levels were significantly increased in
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the group treated with both ATM and PTEN. When the ATM-specific inhibitor KU55933 was used to inhibit ATM activity, the p-PTEN (Ser113) levels were decreased. The results indicated that PTEN was directly phosphorylated by ATM.
TPT-induced PTEN translocation is dependent on PTEN (Ser113) phosphorylation To further determine the role of phosphorylation of PTEN (Ser113) in ATM-mediated PTEN nuclear 7
translocation in response to TPT, the mutant plasmid 3×Flag-PTENS113A was constructed. HeLa cells were transiently transfected with the 3×Flag-PTENS113A plasmid. After treatment with TPT, the cells transfected with the 3×Flag-PTEN-WT plasmid showed PTEN nuclear translocation and PTEN Ser113 phosphorylation; However, evidence for PTEN nuclear translocation and PTEN (Ser113) phosphorylation was not observed by confocal microscopy and western blotting in cells with 3×Flag-PTENS113A (Fig. 3A and B). Also, when
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HeLa cells expressing 3×Flag-PTEN-WT or 3×Flag-PTENS113A were treated with CDDP, similar results
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were obtained (Fig. 1D).
When the cells were treated with 10ng/mL nuclear export inhibitor leptomycin B and TPT, PTENS113A
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localized in the nucleus (Fig. 3C). This result indicates that this mutant can enter the nucleus but is not
phosphorylation in A549 and HeLa cells.
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retained there. Therefore, TPT-induced PTEN translocation is dependent on PTEN (Ser113)
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SUMO proteins are small ubiquitin-like modifiers. Similar to protein phosphorylation, SUMOylation is also a post-translational modifier involved in various cellular processes. It was reported that SUMOylation
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of PTEN controls its nuclear localization.25 To investigate if PTEN (Ser113) phosphorylation was involved
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in PTEN SUMOylation, HeLa cells carrying 3×Flag-PTEN-WT or 3×Flag-PTENS113A were transfected with HA-SUMO1 and treated with 3 μg/mL TPT for 24 h. HA-immunoprecipitates (IP) were immunoblotted for
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SUMO-PTEN. As shown in Fig. S2, SUMO-PTEN was observed, and its level was increased upon TPT
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treatment in both cells expressing PTEN-WT and PTENS113A. The result suggests that Ser113 phosphorylation is not involved in PTEN SUMOylation.
Mutant PTENS113A enhances TPT-induced DNA damage An initial step in the mammalian cell response to DNA double-strand breaks is the phosphorylation of histone H2AFX at serine 139 at the site of DNA breaks. Once ATM is activated at a DNA double-strand 8
break, it immediately phosphorylates histone H2AFX at the site of the break, thereby signaling to the cell that a DSB has occurred.26 Formation of phosphorylated H2AFX at Ser139 (termed γ-H2AFX), is a rapid and sensitive cellular response to the presence of DSBs.27 TPT is a specific inhibitor of topoisomerase I in the nucleus. Immunofluorescence and confocal microscopy were used to assess whether TPT induces DNA damage in A549 and HeLa cells. A549 and HeLa cells were treated with 3 μg/mL TPT for 24 h. g-H2AFX
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foci were observed in TPT-treated cells after 12 h and 24 h and increased in a time-dependent manner,
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whereas no detectable foci was observed in the control group (Fig. S3A). Bar graphs representing the percentage of g-H2AFX-positive cells at different time-points were presented in Fig. S3B.
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Upon treatment with 3 μg/mL TPT for 0 h to 36 h, the expression of γ-H2AFX protein increased in a
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time-dependent manner in A549 and HeLa cells (Fig. S3C). The results indicate that TPT induces DBS in A549 and HeLa cells.
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We discovered that phospho-PTEN (Ser113) plays a key role in PTEN nuclear translocation. To further determine the role of PTEN (Ser113) in TPT-induced DNA damage, A549 and HeLa cells were transiently
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transfected with the 3×Flag-PTEN-WT or 3×Flag-PTENS113A plasmid 24 h prior to 3 μg/mL TPT treatment
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for 24 h. As shown in Fig. 4A, g-H2AFX foci were notably increased in the cells with the 3×FlagPTENS113A plasmid compared with the cells with the 3×Flag-PTENS113A plasmid following TPT treatment.
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4B).
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Bar graph represents percentage of cells with g-H2AFX-positive cells in control or TPT-treated for 24 h (Fig.
The western blotting results indicated that the g-H2AFX expression levels were enhanced in all
TPT-treated groups, with a remarkable increase noted with the 3×Flag-PTENS113A plasmids. These results are consistent with the immunofluorescence results (Fig. 4C), suggesting that mutant PTENS113A enhances TPT-induced DNA damage in A549 and HeLa cells. The same results were obtained using A549 cells with PTEN shRNA-mediated knockdown (Fig. S3D) 9
Moreover, since DSB repair is affected by the cell cycle stage, we sought to determine whether the PTENS113A mutation affected the cell cycle stage using flow cytometry. A549 cells were transiently transfected with the 3×Flag-PTEN-WT or 3×Flag-PTENS113A plasmid 24 h prior to TPT treatment. As shown in Fig. S4A, the percentage of cells in G0/G1 phase increased in the vector, PTEN-WT and PTENS113A group, with no significant difference between the PTEN-WT and PTENS113 group.
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TPT-stimulated apoptosis has been reported in S-phase A549 cells.28 To investigate if PTENS113A
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mutant affect cell apoptosis, we examinated the expression level of cleaved CASP3/caspase 3 and PARP (poly [ADP-ribose] polymerase 1) protein. The cleaved CASP3 and PARP levels were increased with 24 h
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TPT treatment in all the groups. However, the increase in the expression levels of cleaved CASP3 and PARP
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were more significant in cells with 3×Flag-PTEN-WT compared to those expressing 3×Flag-PTENS113A upon 48 h TPT treatment (Fig. S4B). These results suggested that the PTENS113A mutation did affect cell
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apoptosis and further confirmed that PTEN nuclear translocation played a key role in its tumor-suppressor
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function.
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Nuclear translocation of PTEN regulates TPT-induced autophagy The microtubule-associated protein 1 light chain 3 (MAP1LC3/LC3), an ortholog of yeast Atg8, exists
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on the autophagosome and phagophore membranes. MAP1LC3-II/LC3-II (the cleaved and lipidated form of
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microtubule-associated protein 1) amounts correlate well with the number of autophagosomes.29 Enhancement of the conversion of MAP1LC3-I/LC3-I (soluble form of microtubule-associated protein 1 light chain 3) to MAP1LC3-II/LC3-II and upregulation of MAP1LC3/LC3 expression occurs when autophagy is induced.30 SQSTM1/p62 possesses a short region that interacts with MAP1LC3/LC3.31 SQSTM1/p62 participates in autophagy and is degraded in the autolysosome. MAP1LC3-II/LC3-II and SQSTM1/p62 are reliable markers of autophagy. To test whether TPT induces autophagy in cancer cells, we 10
first assessed YFP-LC3 accumulation and punctate formation using confocal microscopy. YFP-LC3 plasmids were transiently transfected into A549 and HeLa cells for 24 h, and the cells were then treated with 3 μg/mL TPT for an additional 24 h. YFP-LC3 significantly accumulated in the cytoplasm, which suggested autophagosome formation (Fig. 5A). Next, western blot analysis revealed that MAP1LC3-II/LC3-II expression markedly increased but that level of SQSTM1/p62 significantly decreased due to degradation in a
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concentration- and time-dependent manner in TPT-treated A549 and HeLa cells (Fig. 5B). These results
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suggest that TPT induces the conversion of MAP1LC3-I/LC3-I to MAP1LC3-II/LC3-II and the degradation of SQSTM1/p62 in A549 and HeLa cells undergoing TPT-stimulated autophagy.
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Many studies have demonstrated that PTEN positively regulates autophagy. PTEN is the central
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negative regulator of the PI3K signal transduction cascade. To determine whether PTEN knockdown affects autophagy in A549 and HeLa cells, PTEN shRNA plasmids were stably transfected into these cells. A549
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and HeLa cells were first treated with 3 μg/mL TPT for 24 h, and the expression of MAP1LC3/LC3 and SQSTM1/p62 was analyzed by immunoblotting. As shown in Fig. 6A, MAP1LC3-II/LC3-II expression
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increased in the TPT-treated groups, but MAP1LC3-II/LC3-II expression was reduced in cells transfected
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with the PTEN shRNA plasmid. At the same time, the SQSTM1/p62 level decreased due to degradation in the TPT-treated groups, but the effect was partly reversed when cells were transfected with the PTEN
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shRNA plasmid. Bar graphs represented the relative MAP1LC3-II/LC3-II protein levels normalized to that
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of GAPDH of different groups.
Next, we sought to determine whether PTEN overexpression regulates autophagy. A549 and HeLa cells
were transiently transfected with the 3×Flag-PTEN-WT plasmids for 24 h and treated with 3 μg/mL TPT for an additional 24 h. As shown in Fig. 6B, Flag and PTEN overexpression was detected in the 3×Flag-PTEN-WT group, and MAP1LC3-II/LC3-II expression was significantly increased in all TPT-treated groups. A relatively high level of MAP1LC3-II/LC3-II was detected in the 3×Flag-PTEN-WT + 11
TPT group in both A549 and HeLa cells. SQSTM1/p62 protein degradation was detected in all TPT-treated groups, and a relatively low level of SQSTM1/p62 was detected in the 3×Flag-PTEN-WT + TPT group in both A549 and HeLa cells. Our results demonstrate that PTEN regulates TPT-induced autophagy in A549 and HeLa cells. The same results were obtained using A549 cells with PTEN shRNA-mediated knockdown. As shown in Fig. S5A, increased MAP1LC3-II/LC3-II expression and SQSTM1/p62 degradation were
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observed in cells with 3×Flag-PTEN-WT (shRNA-resistant) after TPT treatment. Bar figures represented the
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relative MAP1LC3-II/LC3-II protein levels normalized to that of GAPDH of different groups.
Because PTEN (Ser113) phosphorylation could affect PTEN translocation, we further assessed the
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relationship between PTEN Ser113 phosphorylation and TPT-induced autophagy in A549 and HeLa cells.
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A549 and HeLa cells were transiently transfected with vector and the 3×Flag-PTEN-WT and 3×Flag-PTENS113A plasmids and treated with TPT for an additional 24 h. The conversion of
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MAP1LC3-I/LC3-I to MAP1LC3-II/LC3-II was detected in all TPT-treated groups. MAP1LC3-II/LC3-II expression was increased in the cells with 3×Flag-PTEN-WT compared with the cells with
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3×Flag-PTENS113A after TPT treatment. SQSTM1/p62 protein degradation was detected in all TPT-treated
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groups, and a relatively low level of SQSTM1/p62 was detected in the 3×Flag-PTEN-WT + TPT group in both A549 and HeLa cells (Fig. 6C). The same results were obtained using A549 cells with PTEN
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shRNA-mediated knockdown. As shown in Fig. S5B, increased MAP1LC3-II/LC3-II expression and
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SQSTM1/p62 degradation were observed in cells with 3×Flag-PTEN-WT (shRNA-resistant) compared with those in the cells expressing 3×Flag-PTENS113A (shRNA-resistant) after TPT treatment. Immunofluorescence and confocal microscopy were used to assess the relationship between PTEN
Ser113 phosphorylation and TPT-induced autophagy in HeLa cells. YFP-LC3 plasmids were transiently transfected into HeLa cells. As shown in Fig. 6D, YFP-LC3 staining was notably increased in the cells with the 3×Flag- PTENS113A plasmid compared with those in cells with 3×Flag-PTENS113A plasmid following 12
TPT treatment. Bar graphs represented the percentage of YFP-LC3-positive HeLa cells of different groups. Moreover,
in
A549
and
HeLa
cells
transfected
with
the
3×Flag-PTEN-WT
plasmid,
MAP1LC3-II/LC3-II accumulation was markedly increased in the presence of bafilomycin A1 (Baf.A1), a lysosomal protease
inhibitor, confirming that
TPT
activates autophagic flux.32 By contrast,
MAP1LC3-II/LC3-II formation was slightly decreased in A549 and HeLa cells transfected with the
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3×Flag-PTENS113A plasmid, SQSTM1/p62 protein degradation was detected in all TPT-treated groups, and a
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relatively low level of SQSTM1/p62 was detected in the 3×Flag-PTEN-WT + TPT group in both A549 and HeLa cells (Fig. 6E). The same results were obtained using A549 cells with PTEN shRNA-mediated
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knockdown. As shown in Fig. S5C, increased MAP1LC3-II/LC3-II expression and SQSTM1/p62
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degradation were observed in cells with 3×Flag-PTEN-WT (shRNA-resistant) compared with those in the cells expressing 3×Flag-PTENS113A (shRNA-resistant) after TPT treatment. MAP1LC3-II/LC3-II
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accumulation was markedly increased in the presence of Baf.A1 in all groups. The results indicated that PTEN phosphorylation at Ser113 is required for autophagy.
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Next, we want to determine how PTEN phosphorylation and nuclear translocation contribute to
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autophagic induction. JUN-AMPK is considered as a “stress-activated protein kinase” pathway and plays an essential role in autophagy. We have previously demonstrated that MAPK8/JNK1-MAPK9/JNK2 induces
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the transcriptional activation of SESN2 (sestrin2), a novel player in autophagy induction given its role in
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AMPK (protein kinase, AMP-activated) activation, via phosphorylation of JUN (jun proto-oncogene).33 Therefore, we assessed the phosphorylated proteins (JUN, AMPK, RPS6KB/p70S6K) and SESN2 levels in the cells with vector or 3×Flag-PTEN-WT or 3×Flag-PTENS113A after TPT treatment. Increased p-JUN-SESN2-p-AMPK and reduced p-RPS6KB expression were observed in the cells with 3×Flag-PTEN-WT compared with the cells with 3×Flag-PTENS113A following TPT treatment (Fig. 7A). The same results were obtained using A549 cells with PTEN shRNA-mediated knockdown (Fig. S6A). JUN or 13
AMPK siRNA respectively inhibited JUN or AMPK activation. A549 cells and HeLa cells with reduced JUN or AMPK expression due to siRNA treatment were exposed to 3 mg/mL TPT for 24 h. As shown in Fig. 7B, the p-JUN, p-AMPK and SESN2 levels were significantly enhanced and p-RPS6KB reduced in the TPT treatment group. However, knockdown of JUN by siRNA significantly reduced the p-JUN, p-AMPK and SESN2 protein levels and p-RPS6KB increased. AMPK activation plays a key role in autophagic induction.
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As shown in Fig. 7C, the p-AMPK levels were significantly enhanced and p-RPS6KB reduced in the TPT
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treatment group. Then, knockdown of AMPK markedly decreased the p-AMPK protein levels and p-RPS6KB increased again. At the same time, Flag and PTEN overexpression was detected in the groups,
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and MAP1LC3-II/LC3-II expression was significantly increased in all TPT-treated groups. A relatively high
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level of MAP1LC3-II/LC3-II was detected in the 3×Flag-PTEN-WT + TPT group in both A549 and HeLa cells (Fig. 7B and C). However, there was no difference in phospho-MAPK(8/9)/JNK(1/2) levels in the
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cells with 3×Flag-PTEN-WT or 3×Flag-PTENS113A following TPT treatment (data not shown). The same results were obtained using A549 cells with PTEN shRNA-mediated knockdown (Fig. S6B and C).
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Collectively, these results suggest that PTEN nuclear translocation promotes autophagy via a mechanism
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associated with p-JUN-SESN2-AMPK pathway activation in A549 and HeLa cells. Moreover, many studies have demonstrated that PTEN is a known inhibitor of AKT, which negatively
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regulates autophagy. We sought to determine whether PTEN phosphorylation affected the AKT pathway.
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A549 cells were transiently transfected with vector and the 3×Flag-PTEN-WT and 3×Flag-PTENS113A plasmids and treated with TPT for an additional 24 h. As shown in Fig. 7D, p-GSK3B and p-AKT levels were not different in the cells with vector or 3×Flag-PTEN-WT or 3×Flag-PTENS113A following TPT treatment. The results suggested that PTEN phosphorylation has no effect on the AKT pathway. PTEN inactivation results in the constitutive activation of the PI3K-AKT pathway and a subsequent increase in protein synthesis, cell cycle progression, migration, and survival.4,5 14
Discussion This study demonstrates for the first time that ATM activation by TPT directly phosphorylates PTEN at Ser113 to induce PTEN nuclear translocation. PTEN nuclear translocation triggers autophagy induction through activation of the p-JUN-SESN2-AMPK pathway in response to TPT treatment.
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ATM, a phospholipid acyl enzyme-3-kinase that directly responds to DNA damage, is mutated in the
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disease ataxia telangiectasia. ATM is activated in response to DNA damage, particularly DNA double-strand breaks (DSBs).34 ATM is involved in various cell signaling events, including the acetylation,
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phosphorylation, and activation of proteins related to cell cycle and DNA damage signaling. In this study,
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we found that ATM kinase was activated by TPT or CDDP treatment in a concentration- and time-dependent manner. Also, we found that endogenous and exogenous PTEN translocates to the nucleus after TPT or
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CDDP treatment, and PTEN translocation is clearly suppressed when ATM activity is inhibited by the ATM specific inhibitor KU55933 or ATM siRNA. These results indicate that DNA damaging agents-induced
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PTEN translocation is dependent of ATM kinase activation. In this study, we first identified a possible ATM phosphorylation site at PTEN serine 113 (Ser113)
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using Scansite software. Next, we generated a phospho-specific antibody against PTEN Ser113.
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Coimmunoprecipitation and l-phosphatase were employed to evaluate the specificity of the phospho-PTEN Ser113 antibody. In addition, we found that p-PTEN (Ser113) levels increased in a dose-dependent manner
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in A549 cells and HeLa cells after TPT or CDDP exposure. When ATM expression was reduced by ATM siRNA, TPT-induced PTEN (Ser113) phosphorylation was inhibited. To illustrate the role of PTEN (Ser113) phosphorylation in PTEN nuclear translocation, we transfected the mutated 3×Flag-PTENS113A plasmid and the wild-type 3×Flag-PTEN-WT plasmid into HeLa cells. After treatment with TPT, we found that PTEN did not translocate to the nucleus in the cells harboring the 3×Flag-PTENS113A plasmids. Furthermore，the in vitro phosphorylation assay with purified proteins demonstrated that PTEN was directly phosphorylated by ATM. 15
PTEN activity, localization and stability are regulated by acetylation, ubiquitination, oxidation, and phosphorylation. CSNK2/CK2 (casein kinase 2) primarily phosphorylates PTEN at Ser370 and Ser385, and GSK3B (glycogen synthase kinase 3 beta) phosphorylates PTEN at Ser362 and Thr366.35,36 GLTSCR2/PICT-1 (glioma tumor suppressor candidate region 2) promotes PTEN phosphorylation at Ser380.37 ROCK (RhoA-associated, coiled-coil containing kinase) phosphorylates PTEN at Ser229, Thr232,
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Thr319 and Thr321 in the C2 domain to activate and target PTEN to the plasma membrane.38
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Phospho-PTEN typically refers to PTEN characterized by cytoplasmic localization, enhanced stability, lower activity, and a closed structure. Our findings indicate that Ser113 is a new PTEN phosphorylation site
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that promotes PTEN nuclear translocation. SUMOylation is also a post-translational modification involved
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in various cellular processes. Bassi C et al. report that SUMOylation of PTEN controlled its nuclear localization. They indicate that unlike wild-type SUMO-PTEN, which can rapidly be excluded from the
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nucleus, the SUMO-PTENS/T398A mutant cannot exit the nucleus in cells exposed to ionizing radiation.25 In our study, PTEN-WT and PTENS113A were SUMOylated upon treatment with TPT. However, when we
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treated cells with nuclear-export inhibitor Leptomycin B, our results indicated that the PTENS113A mutant
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could enter the nucleus but could not be retained there. It suggested that ATM-mediated PTEN (Ser113) phosphorylation was not involved in PTEN SUMOylation. Our findings indicate that Ser113 is a new PTEN
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phosphorylation site that promotes PTEN nuclear translocation.
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As a tumor suppressor, nuclear PTEN may also maintain normal chromosomal structure and function. Based on the nuclear functions of PTEN, we speculate that ATM-mediated PTEN nuclear translocation affects DNA damage signaling. Using g-H2AFX staining as an indicator of DSBs, we found that TPT-induced DNA damage signaling is perturbed in A549 cells and HeLa cells. Moreover, inhibition of PTEN nuclear translocation increases the number of cells with g-H2AFX foci staining. Our findings reveal that PTEN nuclear translocation, which is regulated by phosphorylation of PTEN at Ser113, is required to 16
repair DNA damage. This finding is consistent with previous studies. Moreover, DNA break-repair is affected by the cell cycle stage. TPT-stimulated apoptosis has been reported in S-phase A549 cells.28 In this study, we found that PTENS113A mutation had no effect on cell cycle stage and cell apoptosis. Autophagy is characterized by the sequestration and delivery of bulk cytoplasm and organelles in double or multimembrane autophagic vesicles for subsequent degradation by the cell’s own lysosomal
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system.39 During the formation of autophagosomes, MAP1LC3/LC3 is lipidated. The subsequent
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LC3-phospholipid conjugate (MAP1LC3-II/LC3-II) is localized to autophagosomes and autolysosomes, and MAP1LC3-II/LC3-II accumulation is used as a marker of autophagy.29 Furthermore, after PTEN nuclear
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translocation, levels of cytoplasmic PTEN are hypothesized to decrease, leading to PI3K-AKT pathway
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activation and sequential inhibition of autophagic induction. In contrast, we found that autophagic induction significantly increased in the cells harboring the 3×Flag-PTEN-WT plasmid compared with the
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3×Flag-PTENS113A plasmid. This indicated that PTEN nuclear translocation promotes autophagic induction following TPT treatment.
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How does PTEN nuclear translocation contribute to autophagic induction? SESN2 is a TP53 target gene
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that activates the AMPK pathway to induce autophagy. However, TP53 is mutated and nonfunctional in A549 and HeLa cells; thus, TP53 is not involved in the regulation of SESN2 expression in these 2 cell lines.
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We have previously demonstrated that activated JUN activates SESN2 expression at the transcriptional level
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through binding to the promoter of SESN2, potentially activating AMPK to induce autophagy through inhibition of MTORC1 (mechanistic target of rapamycin complex 1) in colon cancer cells.31 Here, we found that SESN2 and p-AMPK expression increased and p-RPS6KB expression reduced in the cells with 3×Flag-PTEN-WT following TPT treatment. JUN is a member of the Jun family that consists of JUN, JUNB, and JUND. JUN is a component of the transcription factor activator protein-1 (AP-1).40 In addition, Dunn et al. report that JUN is a principal target of MAPK(8/9)/JNK(1/2). In this study, p-JUN (Ser63), p-AMPK and 17
SESN2 protein levels were relatively increased and p-RPS6KB reduced in the cells harboring 3×Flag-PTEN-WT compared with the cells harboring 3×Flag-PTENS113A after TPT treatment. A relatively high level of MAP1LC3-II/LC3-II was detected in the 3×Flag-PTEN-WT + TPT group in both A549 and HeLa cells. However, p-MAPK(8/9)/JNK(1/2) expression did not vary between the 3×Flag-PTEN-WT + TPT and 3×Flag-PTENS113A + TPT groups (data not show). Therefore, we speculate that PTEN nuclear
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translocation regulates JUN phosphorylation and stability. The level of JUN phosphorylation at the
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C-terminal sites Thr239 and Ser243 has been reported to participate in its downregulation.41 Thus, whether nuclear PTEN affects JUN (Ser243) phosphorylation via its phosphatase activity is of interest.
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In conclusion, the results presented here provide evidence that ATM regulates PTEN phosphorylation at
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Ser113, as a result of exposure to DNA-damaging agents, and promotes PTEN nuclear translocation, which further promotes autophagy through activation of the p-JUN-SESN2-AMPK pathway in tumor cells. We
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conclude that autophagy requires PTEN nuclear translocation that is regulated by PTEN (Ser113)
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Cell culture
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Materials and Methods
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phosphorylation in response to DNA-damaging agent treatment in cancer cells.
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The human cervical cancer (HeLa) and lung adenocarcinoma (A549) cell lines were obtained from American Type Culture Collection (CCL-2 and CRM-CCL-185, respectively) and have been stored in liquid nitrogen at our lab. Both cell lines were cultivated in DMEM and RMPI 1640 medium, respectively, supplemented with 10% fetal bovine serum, penicillin (100 μg/mL) and streptomycin (100 μg/mL) in a 5% CO2 humidified atmosphere at 37 ºC. All experiments were performed in these cells between passage 10 and 20 at the confluency of 70%. 18
Drugs and reagents Topotecan (TPT; 004) was purchased from GlaxoSmithKline. KU55933 (587871-26-9) was purchased from Millipore. RPMI-1640 medium (31800-022), DMEM medium (12800-017), fetal bovine serum, the anti-flag antibody, anti-TUBA4A (t6199) and ATP (A1582) were obtained from Sigma Aldrich.
(5883),
anti-HIST1H3A/histone
H3
(9715),
anti-RPS6KB/p70S6K
(2708),
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phospho-ATM
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Anti-SQSTM1/p62 (8025), phospho-H2AFX (γ-H2AFX; 9718), anti-PTEN (9559), anti-ATM (2873),
phospho-RPS6KB/p70S6K (9234) and anti-normal mouse IgG (7076) were purchased from Cell Signaling
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Technology. The anti-MAP1LC3/LC3 antibody (NB100-2220) was obtained from Novus Biologicals.
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Anti-phospho-PTEN antibody was prepared by the Abgent, Inc. Lipofectamine 2000 (11668-027), goat anti-rabbit Alexa 488 (997773) and opti-MEM medium (22600050) were purchased from the InvitrogenTM.
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Purified ATM protein (active) (14-933) was purchased from Millipore, and purified PTEN recombinant protein (TP710023) was obtained from Origene. ATM siRNA, JUN siRNA and AMPK siRNA were obtained
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from GenePharma Co, Ltd. The 3×Flag-PTEN-WT plasmid and the 3×Flag-PTENS113A plasmid were
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(S1726).
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prepared by Fulen Gene Company. Leptomycin B was purchased from Beyotime Institute of Biotechnology
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Immunoblotting analysis
A549 and HeLa cells were harvested and lysed in 100 μL of lysis buffer (20 mM Tris-HCl, pH 7.5, 150
mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100 (Sigma-Aldrich, 234729), 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin) after treatment with TPT for the indicated times. Lysates were centrifuged at 13,400 g for 15 min at 4 ºC. The supernatant fraction was then collected. Protein concentrations were determined using the Pierce BCA protein assay kit (Thermo, 19
1862634). Whole cell lysates were prepared by adding 6×SDS-PAGE buffer (Serva, 151-21-3) and incubation at 100 ºC for 10 min for protein denaturation. Equivalent aliquots of protein samples (20 to 40 μg) were loaded and electrophoresed on SDS-PAGE gels and then transferred to polyvinylidene fluoride/PVDF membranes. The membranes were first blocked with 5% nonfat dry milk for 60 min at room temperature and then incubated with primary antibodies overnight at 4 ºC. Next, the blots were incubated with horseradish
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peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, sc-2004 and sc-2005) for 1 h at room
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temperature. After 3 washes with TBST (10 mM Tris, 150 mM NaCl, pH 8.0, 0.1% Tween 20 [MP Biomedicals, 103168]) for 5 min, the proteins were visualized with the ECL chemiluminescence reagent and
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exposure to radiograph film.
Immunofluorescence
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A549 and HeLa cells were seeded in 24-well plates containing NUNC Thermanox™ coverslips. Upon treatment, cells were fixed with a 4% paraformaldehyde fixative solution at room temperature for 20 min
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and washed 3 times in 1×PBS (0.8% NaCl, 0.02% KCl, 0.144% Na2HPO4, 0.024% KH2PO4, pH7.4) for 5
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min each. Cells were then permeabilized with 0.25% Octoxynol 9/Triton X-100 (Sigma-Aldrich, 9002-93-1) in PBS at 4 °C for 10 min and blocked with 4% BSA (MP Biomedicals, 0218054991) blocking solution at
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37 ºC for 30 min. The cells were incubated with the primary antibody (1:300) overnight at 4 ºC. Cells were
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washed thrice in PBST for 5 min and incubated with the fluorescent secondary antibody at room temperature for 1 h. DAPI was added for 5 min to visualize nuclei. After using an antifluorescence quenching reagent, the coverslips were stored at 4 ºC. Cells were observed using a laser scanning confocal microscope (Olympus, FV-1000).
shRNA and siRNA transfection The target sequence for ATM or JUN or AMPK siRNA was respectively 5'-AAGGTGAA 20
GATATATTCCTCC-3',5'-UGCCUACCAUCUCAUAAUAtt-3', 5′-AGAUGG AAACGACCUUCUATT-3′. The hairpin RNA (shRNA) sequence targeting PTEN was 5'-GATCTTGACCAATGGCTAAGT-3'. The control siRNA sequence was 5'-UUC UCC GAA CGU GUC ACG UTT-3' and synthesized by GenChem Co. HeLa and A549 cells were seeded into 6-well plates the day before transfection. When the cells reached 40% to 60% confluence, transfection was performed using serum-free Opti-MEM media (Gibco, 31985-070), lipofectamine 2000 (Invitrogen, 11668-019) and siRNA according to the manufacturer’s recommendations.
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The medium was replaced with fresh growth medium containing 10% fetal bovine serum 6 h after
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transfection, and cells were incubated for an additional 24 h for subsequent experiments.
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In vitro phosphorylation assay
This assay was carried out using commercially available active ATM kinase (1 μg) and PTEN
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recombinant protein (1 μg). Reaction was initiated by adding ATP and carried out at 30 °C in 25 μL kinase buffer [20 mM Tris·HCl (pH 7.5), 2 mM EDTA, 10 mM MgCl2, 1 mM DTT] for 30 min. The reaction was
Cell cycle analysis
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terminated by adding 12.5 μL 3x loading buffer and incubating at 100 ºC for 10 min.
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Cell cycle analysis was carried out by flow cytometry. Briefly, A549 cells were seeded into 6-well culture plates, treated as indicated, collected, fixed, stained with propidium iodide (1 μg/mL) and flow
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cytometric analysis on a FACScan with a 488 nm Argon laser (Backman Coulter, FC500). Quantitation was
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performed by scoring cells for sub-G0/G1 DNA content using the SigmaPlot software.
In vitro SUMOylation HeLa cells were transiently transfected with 10 μg of the HA-SUMO1 and 10 μg 3×Flag-PTEN-WT or 3×Flag-PTENS113A plasmid. Equal amounts of protein sample were incubated with a flag monoclonal antibody for 2 h at 4 ºC. The antibody-protein complex was mixed with protein A Sepharose 4 fast flow 21
solution for 16 h at 4 ºC. After centrifugation, the pellet fraction was washed 4 times and resuspended in loading buffer. The suspension was boiled for 10 min at 100 ºC, centrifuged and analyzed by SDS–PAGE and western blotting with anti-HA antibody.
Plasmids and transfection
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The vector pcDNA3.1 and YFP-LC3 plasmids were kindly gifted by Dr. Rong Deng, Dr. Dandan Li
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and Dr. Xiaoqi Wu, Cancer Center, Sun Yat-sen University, Guangzhou, China. The 3×Flag-PTEN-WT and 3×Flag-PTENS113A, 3×Flag-PTEN-WT (shPTEN-resistant) and 3×Flag-PTENS113A (shPTEN-resistant)
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plasmids were produced by the Gene Copoeia Company. HA-SUMO1 plasmid was purchased from
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Addgene (plasmid 21154). All mutations were verified by DNA sequencing. Transfection was performed as
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previously described.42,43
Cytoplasmic and nuclear protein extraction
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Cytosolic and nuclear fractions were prepared using a nuclear-cytosolic protein isolation kit (Beyotime Institute of Biotechnology). HeLa and A549 cells were seeded in 10 cm plates. After treatment with 3
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mg/mL TPT for 24 h, cells were collected and washed with 1 mL of PBS and then centrifuged in 4 ºC at 90 g
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for 3 min. The resultant supernatant fraction was discarded, and the sediment was resuspended in reagent A
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with fresh PMSF. Samples were vortexed vigorously for 5 sec and placed on ice for 15 min before adding reagent B. The samples were centrifuged at 13,400 g for 3 min at 4 ºC. The supernatant fraction containing the cytosolic proteins was collected and used immediately or stored at -80 ºC. The sediment containing the nuclear proteins was resuspended in nuclear protein extraction reagent with PMSF. The samples were vortexed at 13,400 g for 30 sec, placed on ice for 2 min, and vortexed for 30 sec. The last 2 steps were repeated for 30 min, and the samples were centrifuged at 13,400 g for 10 min at 4 ºC. The supernatant
22
fractions containing the nuclear proteins was used immediately or stored at -80 ºC. The concentrations of the cytosolic and nuclear proteins were measured using the bicinchoninic acid/BCA method (Thermo, 1862634).
Coimmunoprecipitation
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A549 and HeLa cells were transiently transfected with the 3×Flag-PTEN-WT plasmid. Cells were lysed
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with RIPA lysis buffer, and the protein concentration for each group was measured using the bicinchoninic acid method. Equal amounts of protein sample were incubated with a flag monoclonal antibody for 2 h at 4
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ºC. The antibody-protein complex was mixed with Protein A/G Agarose (Roche, 11243233001) for 16 h at 4
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ºC. After centrifugation, the pellet fraction was washed 4 times and resuspended in loading buffer. The suspension was boiled for 10 min at 100 ºC and centrifuged, and the supernatant fraction was used for
Statistical analysis
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western blotting to identify the coprecipitated proteins.
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All assays were performed in triplicate. Data are expressed as the mean ± standard deviation (SD). Statistical analyses were performed with the 2-tailed Student t test for comparison of 2 groups by SPSS 13.0
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software. P
Jing-Hong Chen,1,2,† Peng Zhang,4,† Wen-Dan Chen,1,† Dan-Dan Li,1 Xiao-Qi Wu,5 Rong Deng,1 Lin Jiao,1
State Key Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine; Cancer Center;
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1.
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Xuan Li,1 Jiao Ji,1 Gong-Kan Feng,1 Yi-Xin Zeng,1 Jian-Wei Jiang,3* and Xiao-Feng Zhu 1*
Sun Yat-sen University; Guangzhou, China
Department of Hematology; The Second Affiliated Hospital; Guangzhou Medical University; Guangzhou, China
3
Department of Biochemistry; The School of Medicine; Jinan University; Guangzhou, China
4
Department of Microbiology and Immunology; The School of Medicine; Jinan University; Guangzhou, China
5
Department of Science and Research; The Third Affiliated Hospital; Sun Yat-sen University; Guangzhou, China
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2
† These authors contributed equally to this work
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*Correspondence to: Xiao-Feng Zhu; Email: [email protected]; Jian-Wei Jiang; Email:[email protected]
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Keywords: ATM, autophagy, DNA damage, PTEN, topotecan Abbreviations: AKT/PKB, v-akt murine thymoma viral oncogene homolog; AMPK, protein kinase,
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AMP-activated; ATG, autophagy-related; ATM, ATM serine/threonine kinase; Baf.A1, bafilomycin A1;
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CASP3, caspase 3, apoptosis-related cysteine peptidase; CCND1, cyclin D1; CDDP, cisplatin; CENPC/CENP-C, centromere protein C; CITED1/p300/CBP, Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 1; CSNK2/CK2, casein kinase 2; DSBs, DNA double-strand breaks; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GLTSCR2/PICT-1, glioma tumor suppressor candidate region gene 2; GSK3B, glycogen synthase kinase 3 beta; GST, glutathione S-transferase; H2AFX, H2A histone family, member X; JUN, jun proto-oncogene; 1
MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MTORC1, mechanistic target of rapamycin complex 1; MVP, major vault protein; NC, normal control; NEDD4, neural precursor cell expressed, developmentally down-regulated 4, E3 ubiquitin protein ligase; PAGE, polyacrylamide gel electrophoresis; PARP, poly (ADP-ribose) polymerase 1; PI3K, phosphoinositide 3-kinase; PMSF, phenylmethanesulfonyl fluoride; PPase, protein phosphatase; PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5)-trisphosphate; PTEN,
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phosphatase and tensin homolog; RAD51, RAD51 recombinase; RPS6KB/p70S6K; ribosomal protein S6
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kinase, 70kDa; SDS, sodium dodecyl sulfate; SESN2, sestrin 2; siRNA, small interfering RNA; SQSTM1/p62, sequestosome 1; TP53, tumor protein p53; TPT, topotecan; TUBA4A, tubulin, alpha 4a; WT,
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wild type; YFP, yellow fluorescent protein
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PTEN (phosphatase and tensin homolog), a tumor suppressor frequently mutated in human cancer, has various cytoplasmic and nuclear functions. PTEN translocates to the nucleus from the cytoplasm in response
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to oxidative stress. However, the mechanism and function of the translocation are not completely understood. In this study, topotecan (TPT), a topoisomerase I inhibitor, and cisplatin（CDDP）were employed to induce
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DNA damage. The results indicate that TPT or CDDP activates ATM (ATM serine/threonine kinase), which
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phosphorylates PTEN at serine 113 and further regulates PTEN nuclear translocation in A549 and HeLa cells. After nuclear translocation, PTEN induces autophagy, in association with the activation of the
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p-JUN-SESN2/AMPK pathway, in response to TPT. These results identify PTEN phosphorylation by ATM
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as essential for PTEN nuclear translocation and the subsequent induction of autophagy in response to DNA damage.
2
Introduction PTEN/MMAC1 (phosphatase and tensin homolog) has been identified as a tumor suppressor gene from a variety of cancers that display frequent deletion of its location on human chromosome 10q23, a locus that is highly susceptible to mutation in primary human cancers and cancer cell lines.1 PTEN phosphatidylinositol
(3,4,5)-trisphosphate
(PtdInsP3)
to
phosphatidylinositol
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converts
(4,5)-bisphosphate in the cytoplasm, thereby directly antagonizing the activity of phosphoinositide
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3-kinase (PI3K).2,3 PTEN inactivation results in the constitutive activation of the PI3K-AKT pathway
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and a subsequent increase in protein synthesis, cell cycle progression, migration, and survival.4,5 PTEN is localized to the nucleus and cytoplasm. Early immunohistochemical studies of PTEN
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localization found that the endogenous wild-type PTEN protein is exclusively localized in the cytoplasm of various cancer cell lines.6 Subsequently, multiple reports have identified PTEN nuclear or
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perinuclear PTEN in normal primary neurons and endothelial cells, PTEN-positive primary breast
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carcinoma samples, normal epithelial and follicular thyroid cells, follicular thyroid adenomas, and thyroid carcinomas.7,8 However, the nuclear PTEN immunostaining is relatively weak in thyroid
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carcinomas compared with normal thyroid follicular cells and follicular thyroid adenomas.9
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Although normal islet cells exhibit predominantly nuclear PTEN immunostaining in pancreatic tissue, 19 of 23 sporadic endocrine pancreatic tumors predominantly display cytoplasmic PTEN expression patterns.10
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Whiteman observes a similar phenomenon, finding increased nuclear PTEN expression in normal melanocytes and reduced nuclear PTEN expression in melanoma cells. However, PTEN protein expression is primarily localized to the cytoplasm of metastatic melanoma samples. An association between the lack of nuclear PTEN and mitotic index has been reported; nuclear PTEN is evident in quiescent cells, but PTEN primarily localizes to the cytosol of actively dividing cells.11 Generally, PTEN is primarily localized to the nucleus in normal quiescent tissues, whereas cytoplasmic PTEN is predominately found in neoplastic 3
tissues. Thus, nuclear PTEN appears to be essential for tumor suppression. Despite the absence of a classic nuclear localization signal, PTEN enters the nucleus by several mechanisms, including simple diffusion, export dependent on a putative cytoplasmic localization signal, and active shuttling by the RAN GTPase or MVP (major vault protein).12-15 PTEN monoubiquitination at Lys13 or Lys289 by NEDD4 increases its nuclear localization, whereas polyubiquitination by NEDD4 promotes its
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cytoplasmic degradation.16,17
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Accumulating evidence suggests multiple mechanisms of PTEN nuclear import depending on the cell type and cellular environment. Moreover, the role of nuclear PTEN is not the same as that of cytoplasmic
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PTEN. Nuclear PTEN plays a crucial role in regulating cellular homeostasis and stability by downregulating
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the MAPK signal pathway, reducing CCND1 (cyclin D1) levels and G0-G1 arrest, upregulating RAD51 (RAD51 recombinase) levels and double-strand-break repair, and interacting with CENPC/CENP-C
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(centromere protein C), which specifically enhances centromere stability and overall genomic stability by promoting CITED1/p300/CBP(Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal
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domain, 1)-mediated acetylation of TP53/p53 (TRP53 in mice) in response to DNA damage and
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apoptosis.18-23
In this study, we find that ATM (ATM serine/threonine kinase) phosphorylates PTEN at serine 113,
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further promoting PTEN nuclear translocation. PTEN nuclear translocation induces autophagy through
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activation of the p-JUN-SESN2-AMPK pathway following treatment with the DNA-damaging agent topotecan (TPT) in A549 and HeLa cells.
Results
DNA-damaging agents induced ATM phosphorylation 4
TPT, which causes DNA damage, is commonly used for cancer treatment in the clinic. CDDP (cisplatin) is also an anticancer drug that causes cross-linking of deoxyribonucleic acid. ATM kinase is activated in response to DNA double-strand breaks (DSBs). To investigate whether TPT or CDDP treatment induces ATM phosphorylation in A549 and HeLa cells, we determined the levels of phosphorylated ATM by immunoblotting. As shown in Fig. S1, ATM phosphorylation increased in a dose- and time-dependent
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manner after treatment with various concentrations of TPT or CDDP for 24 h and with 3 mg/mL TPT or 6
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phosphorylation and activation in A549 and HeLa cells.
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mg/mL CDDP for various times, respectively. These data indicated that TPT and CDDP induced ATM
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ATM regulates PTEN nuclear translocation, following exposure to DNA-damaging agents It has been previously reported that PTEN accumulates in the nucleus of cells treated with apoptotic
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stimuli, including etoposide, doxorubicin,20 or H2O2, indicating that oxidative stress promotes PTEN nuclear accumulation.24 PTEN is normally found in the cytoplasm in A549 and HeLa cells. After A549 and HeLa
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cells were treated with 3 mg/mL TPT for 24 h, endogenous PTEN translocated from the cytoplasm to the
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nucleus. However, when A549 and HeLa cells were treated with 3 mg/mL TPT and 10 mM KU55933 (ATM specific inhibitor) for 24 h, PTEN nuclear translocation was clearly suppressed (Fig. 1A).
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To detect exogenous PTEN nuclear translocation, the 3×Flag-PTEN-WT plasmid was constructed. The
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3×Flag-PTEN-WT expression vector was transiently transfected into HeLa cells, and the cells were treated with TPT (3 μg/mL) and/or KU55933 (10 μM) for 24 h. The transfection and expression efficiency were determined by immunoblotting (Fig. 1B). When HeLa cells were treated with 3 mg/mL TPT for 24 h, exogenous PTEN translocated from the cytoplasm to the nucleus, consistent with the endogenous PTEN translocation pattern observed by confocal microscopy under the same conditions. When the ATM specific inhibitor KU55933 or transient transfection with ATM siRNA was used to inhibit ATM activity, the PTEN 5
translocation was clearly suppressed (Fig. 1C). Also, when HeLa cells were treated with 6 mg/mL CDDP for 24 h, exogenous PTEN translocated from the cytoplasm to the nucleus, which was consistent with the result from TPT treatment (Fig. 1D). To further confirm PTEN nuclear translocation, nuclear and cytoplasm proteins of A549 cells and HeLa cells were extracted, and the PTEN expression in these components was detected by western blotting.
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As expected, PTEN levels were increased in the nuclear fraction compared with the cytosolic fraction in the
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TPT-treated group, suggesting that TPT induces PTEN nuclear translocation. However, TPT-induced PTEN nuclear translocation was significantly inhibited upon treatment with KU55933 and ATM siRNA (Fig. 1E
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and F), indicating that PTEN nuclear translocation is regulated by ATM.
ATM regulates PTEN phosphorylation, following exposure to DNA-damaging agents
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Phosphorylation is an important protein posttranslational modification. Given that we demonstrated that TPT induces ATM phosphorylation and that ATM further regulates TPT-induced PTEN nuclear
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translocation in A549 and HeLa cells, we wanted to determine whether PTEN nuclear translocation was
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regulated by ATM via phosphorylation.
To determine whether ATM directly phosphorylates PTEN, we first analyzed the PTEN sequence using
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Scansite software (http://scansite.mit.edu) and identified a potential ATM phosphorylation site at serine 113
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(Ser113) (Fig. 2A). Next, we developed a phospho-specific antibody against PTEN (Ser113). To validate the Phospho-PTEN (Ser113) antibody, A549 cells were treated with: 1) 3 mg/mL TPT only; 2) 3 mg/mL TPT and l-phosphatase; 3) 3 mg/mL TPT, l-phosphatase, and protein phosphatase (PPase) inhibitors. The phospho-PTEN (Ser113) levels in A549 cells treated with l-phosphatase were significantly decreased compared with the TPT group. The inhibitory effect was partly restored when PPase inhibitors were applied (Fig. 2B). The results confirmed the specificity of the phospho-PTEN (Ser113) antibody. 6
To determine whether TPT induces PTEN phosphorylation at Ser113, we transiently transfected HeLa cells with the 3×Flag-PTEN-WT plasmid. The cells were then exposed to different concentrations of TPT or CDDP for 24 h. The results indicated that the levels of p-PTEN (Ser113) were enhanced in a concentration-dependent manner (Fig. 2C). In addition, A549 or HeLa cells were treated with 3 μg/mL TPT for 24 h, and immunoprecipitation was
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performed using a Flag antibody. The precipitates were subjected to SDS-PAGE and probed with the
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p-PTEN (Ser113) antibody. As shown in Fig. 2D, the p-PTEN (Ser113) levels in the TPT treatment group were significantly increased compared with the control group, indicating that TPT induces PTEN (Ser113)
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phosphorylation.
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Furthermore, we sought to investigate whether TPT-induced PTEN phosphorylation was regulated by ATM. A549 cells and HeLa cells, with reduced ATM expression by siRNA or KU55933, were treated with
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3 mg/mL TPT for 24 h. As shown in Fig. 2E, the p-ATM and p-PTEN (Ser113) levels were significantly increased in the TPT treatment group. However, knockdown of ATM by siRNA or KU55933 significantly
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reduced the p-PTEN (Ser113) levels. These data indicate that ATM regulates TPT-induced PTEN (Ser113)
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phosphorylation.
The in vitro kinase assays with purified proteins was used to assess whether PTEN was directly
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phosphorylated by ATM. As show in Fig. 2F, the p-PTEN (Ser113) levels were significantly increased in
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the group treated with both ATM and PTEN. When the ATM-specific inhibitor KU55933 was used to inhibit ATM activity, the p-PTEN (Ser113) levels were decreased. The results indicated that PTEN was directly phosphorylated by ATM.
TPT-induced PTEN translocation is dependent on PTEN (Ser113) phosphorylation To further determine the role of phosphorylation of PTEN (Ser113) in ATM-mediated PTEN nuclear 7
translocation in response to TPT, the mutant plasmid 3×Flag-PTENS113A was constructed. HeLa cells were transiently transfected with the 3×Flag-PTENS113A plasmid. After treatment with TPT, the cells transfected with the 3×Flag-PTEN-WT plasmid showed PTEN nuclear translocation and PTEN Ser113 phosphorylation; However, evidence for PTEN nuclear translocation and PTEN (Ser113) phosphorylation was not observed by confocal microscopy and western blotting in cells with 3×Flag-PTENS113A (Fig. 3A and B). Also, when
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HeLa cells expressing 3×Flag-PTEN-WT or 3×Flag-PTENS113A were treated with CDDP, similar results
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were obtained (Fig. 1D).
When the cells were treated with 10ng/mL nuclear export inhibitor leptomycin B and TPT, PTENS113A
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localized in the nucleus (Fig. 3C). This result indicates that this mutant can enter the nucleus but is not
phosphorylation in A549 and HeLa cells.
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retained there. Therefore, TPT-induced PTEN translocation is dependent on PTEN (Ser113)
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SUMO proteins are small ubiquitin-like modifiers. Similar to protein phosphorylation, SUMOylation is also a post-translational modifier involved in various cellular processes. It was reported that SUMOylation
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of PTEN controls its nuclear localization.25 To investigate if PTEN (Ser113) phosphorylation was involved
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in PTEN SUMOylation, HeLa cells carrying 3×Flag-PTEN-WT or 3×Flag-PTENS113A were transfected with HA-SUMO1 and treated with 3 μg/mL TPT for 24 h. HA-immunoprecipitates (IP) were immunoblotted for
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SUMO-PTEN. As shown in Fig. S2, SUMO-PTEN was observed, and its level was increased upon TPT
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treatment in both cells expressing PTEN-WT and PTENS113A. The result suggests that Ser113 phosphorylation is not involved in PTEN SUMOylation.
Mutant PTENS113A enhances TPT-induced DNA damage An initial step in the mammalian cell response to DNA double-strand breaks is the phosphorylation of histone H2AFX at serine 139 at the site of DNA breaks. Once ATM is activated at a DNA double-strand 8
break, it immediately phosphorylates histone H2AFX at the site of the break, thereby signaling to the cell that a DSB has occurred.26 Formation of phosphorylated H2AFX at Ser139 (termed γ-H2AFX), is a rapid and sensitive cellular response to the presence of DSBs.27 TPT is a specific inhibitor of topoisomerase I in the nucleus. Immunofluorescence and confocal microscopy were used to assess whether TPT induces DNA damage in A549 and HeLa cells. A549 and HeLa cells were treated with 3 μg/mL TPT for 24 h. g-H2AFX
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foci were observed in TPT-treated cells after 12 h and 24 h and increased in a time-dependent manner,
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whereas no detectable foci was observed in the control group (Fig. S3A). Bar graphs representing the percentage of g-H2AFX-positive cells at different time-points were presented in Fig. S3B.
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Upon treatment with 3 μg/mL TPT for 0 h to 36 h, the expression of γ-H2AFX protein increased in a
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time-dependent manner in A549 and HeLa cells (Fig. S3C). The results indicate that TPT induces DBS in A549 and HeLa cells.
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We discovered that phospho-PTEN (Ser113) plays a key role in PTEN nuclear translocation. To further determine the role of PTEN (Ser113) in TPT-induced DNA damage, A549 and HeLa cells were transiently
ed
transfected with the 3×Flag-PTEN-WT or 3×Flag-PTENS113A plasmid 24 h prior to 3 μg/mL TPT treatment
pt
for 24 h. As shown in Fig. 4A, g-H2AFX foci were notably increased in the cells with the 3×FlagPTENS113A plasmid compared with the cells with the 3×Flag-PTENS113A plasmid following TPT treatment.
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4B).
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Bar graph represents percentage of cells with g-H2AFX-positive cells in control or TPT-treated for 24 h (Fig.
The western blotting results indicated that the g-H2AFX expression levels were enhanced in all
TPT-treated groups, with a remarkable increase noted with the 3×Flag-PTENS113A plasmids. These results are consistent with the immunofluorescence results (Fig. 4C), suggesting that mutant PTENS113A enhances TPT-induced DNA damage in A549 and HeLa cells. The same results were obtained using A549 cells with PTEN shRNA-mediated knockdown (Fig. S3D) 9
Moreover, since DSB repair is affected by the cell cycle stage, we sought to determine whether the PTENS113A mutation affected the cell cycle stage using flow cytometry. A549 cells were transiently transfected with the 3×Flag-PTEN-WT or 3×Flag-PTENS113A plasmid 24 h prior to TPT treatment. As shown in Fig. S4A, the percentage of cells in G0/G1 phase increased in the vector, PTEN-WT and PTENS113A group, with no significant difference between the PTEN-WT and PTENS113 group.
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TPT-stimulated apoptosis has been reported in S-phase A549 cells.28 To investigate if PTENS113A
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mutant affect cell apoptosis, we examinated the expression level of cleaved CASP3/caspase 3 and PARP (poly [ADP-ribose] polymerase 1) protein. The cleaved CASP3 and PARP levels were increased with 24 h
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TPT treatment in all the groups. However, the increase in the expression levels of cleaved CASP3 and PARP
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were more significant in cells with 3×Flag-PTEN-WT compared to those expressing 3×Flag-PTENS113A upon 48 h TPT treatment (Fig. S4B). These results suggested that the PTENS113A mutation did affect cell
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apoptosis and further confirmed that PTEN nuclear translocation played a key role in its tumor-suppressor
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function.
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Nuclear translocation of PTEN regulates TPT-induced autophagy The microtubule-associated protein 1 light chain 3 (MAP1LC3/LC3), an ortholog of yeast Atg8, exists
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on the autophagosome and phagophore membranes. MAP1LC3-II/LC3-II (the cleaved and lipidated form of
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microtubule-associated protein 1) amounts correlate well with the number of autophagosomes.29 Enhancement of the conversion of MAP1LC3-I/LC3-I (soluble form of microtubule-associated protein 1 light chain 3) to MAP1LC3-II/LC3-II and upregulation of MAP1LC3/LC3 expression occurs when autophagy is induced.30 SQSTM1/p62 possesses a short region that interacts with MAP1LC3/LC3.31 SQSTM1/p62 participates in autophagy and is degraded in the autolysosome. MAP1LC3-II/LC3-II and SQSTM1/p62 are reliable markers of autophagy. To test whether TPT induces autophagy in cancer cells, we 10
first assessed YFP-LC3 accumulation and punctate formation using confocal microscopy. YFP-LC3 plasmids were transiently transfected into A549 and HeLa cells for 24 h, and the cells were then treated with 3 μg/mL TPT for an additional 24 h. YFP-LC3 significantly accumulated in the cytoplasm, which suggested autophagosome formation (Fig. 5A). Next, western blot analysis revealed that MAP1LC3-II/LC3-II expression markedly increased but that level of SQSTM1/p62 significantly decreased due to degradation in a
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concentration- and time-dependent manner in TPT-treated A549 and HeLa cells (Fig. 5B). These results
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suggest that TPT induces the conversion of MAP1LC3-I/LC3-I to MAP1LC3-II/LC3-II and the degradation of SQSTM1/p62 in A549 and HeLa cells undergoing TPT-stimulated autophagy.
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Many studies have demonstrated that PTEN positively regulates autophagy. PTEN is the central
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negative regulator of the PI3K signal transduction cascade. To determine whether PTEN knockdown affects autophagy in A549 and HeLa cells, PTEN shRNA plasmids were stably transfected into these cells. A549
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and HeLa cells were first treated with 3 μg/mL TPT for 24 h, and the expression of MAP1LC3/LC3 and SQSTM1/p62 was analyzed by immunoblotting. As shown in Fig. 6A, MAP1LC3-II/LC3-II expression
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increased in the TPT-treated groups, but MAP1LC3-II/LC3-II expression was reduced in cells transfected
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with the PTEN shRNA plasmid. At the same time, the SQSTM1/p62 level decreased due to degradation in the TPT-treated groups, but the effect was partly reversed when cells were transfected with the PTEN
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shRNA plasmid. Bar graphs represented the relative MAP1LC3-II/LC3-II protein levels normalized to that
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of GAPDH of different groups.
Next, we sought to determine whether PTEN overexpression regulates autophagy. A549 and HeLa cells
were transiently transfected with the 3×Flag-PTEN-WT plasmids for 24 h and treated with 3 μg/mL TPT for an additional 24 h. As shown in Fig. 6B, Flag and PTEN overexpression was detected in the 3×Flag-PTEN-WT group, and MAP1LC3-II/LC3-II expression was significantly increased in all TPT-treated groups. A relatively high level of MAP1LC3-II/LC3-II was detected in the 3×Flag-PTEN-WT + 11
TPT group in both A549 and HeLa cells. SQSTM1/p62 protein degradation was detected in all TPT-treated groups, and a relatively low level of SQSTM1/p62 was detected in the 3×Flag-PTEN-WT + TPT group in both A549 and HeLa cells. Our results demonstrate that PTEN regulates TPT-induced autophagy in A549 and HeLa cells. The same results were obtained using A549 cells with PTEN shRNA-mediated knockdown. As shown in Fig. S5A, increased MAP1LC3-II/LC3-II expression and SQSTM1/p62 degradation were
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observed in cells with 3×Flag-PTEN-WT (shRNA-resistant) after TPT treatment. Bar figures represented the
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relative MAP1LC3-II/LC3-II protein levels normalized to that of GAPDH of different groups.
Because PTEN (Ser113) phosphorylation could affect PTEN translocation, we further assessed the
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relationship between PTEN Ser113 phosphorylation and TPT-induced autophagy in A549 and HeLa cells.
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A549 and HeLa cells were transiently transfected with vector and the 3×Flag-PTEN-WT and 3×Flag-PTENS113A plasmids and treated with TPT for an additional 24 h. The conversion of
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MAP1LC3-I/LC3-I to MAP1LC3-II/LC3-II was detected in all TPT-treated groups. MAP1LC3-II/LC3-II expression was increased in the cells with 3×Flag-PTEN-WT compared with the cells with
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3×Flag-PTENS113A after TPT treatment. SQSTM1/p62 protein degradation was detected in all TPT-treated
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groups, and a relatively low level of SQSTM1/p62 was detected in the 3×Flag-PTEN-WT + TPT group in both A549 and HeLa cells (Fig. 6C). The same results were obtained using A549 cells with PTEN
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shRNA-mediated knockdown. As shown in Fig. S5B, increased MAP1LC3-II/LC3-II expression and
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SQSTM1/p62 degradation were observed in cells with 3×Flag-PTEN-WT (shRNA-resistant) compared with those in the cells expressing 3×Flag-PTENS113A (shRNA-resistant) after TPT treatment. Immunofluorescence and confocal microscopy were used to assess the relationship between PTEN
Ser113 phosphorylation and TPT-induced autophagy in HeLa cells. YFP-LC3 plasmids were transiently transfected into HeLa cells. As shown in Fig. 6D, YFP-LC3 staining was notably increased in the cells with the 3×Flag- PTENS113A plasmid compared with those in cells with 3×Flag-PTENS113A plasmid following 12
TPT treatment. Bar graphs represented the percentage of YFP-LC3-positive HeLa cells of different groups. Moreover,
in
A549
and
HeLa
cells
transfected
with
the
3×Flag-PTEN-WT
plasmid,
MAP1LC3-II/LC3-II accumulation was markedly increased in the presence of bafilomycin A1 (Baf.A1), a lysosomal protease
inhibitor, confirming that
TPT
activates autophagic flux.32 By contrast,
MAP1LC3-II/LC3-II formation was slightly decreased in A549 and HeLa cells transfected with the
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3×Flag-PTENS113A plasmid, SQSTM1/p62 protein degradation was detected in all TPT-treated groups, and a
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relatively low level of SQSTM1/p62 was detected in the 3×Flag-PTEN-WT + TPT group in both A549 and HeLa cells (Fig. 6E). The same results were obtained using A549 cells with PTEN shRNA-mediated
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knockdown. As shown in Fig. S5C, increased MAP1LC3-II/LC3-II expression and SQSTM1/p62
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degradation were observed in cells with 3×Flag-PTEN-WT (shRNA-resistant) compared with those in the cells expressing 3×Flag-PTENS113A (shRNA-resistant) after TPT treatment. MAP1LC3-II/LC3-II
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accumulation was markedly increased in the presence of Baf.A1 in all groups. The results indicated that PTEN phosphorylation at Ser113 is required for autophagy.
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Next, we want to determine how PTEN phosphorylation and nuclear translocation contribute to
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autophagic induction. JUN-AMPK is considered as a “stress-activated protein kinase” pathway and plays an essential role in autophagy. We have previously demonstrated that MAPK8/JNK1-MAPK9/JNK2 induces
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the transcriptional activation of SESN2 (sestrin2), a novel player in autophagy induction given its role in
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AMPK (protein kinase, AMP-activated) activation, via phosphorylation of JUN (jun proto-oncogene).33 Therefore, we assessed the phosphorylated proteins (JUN, AMPK, RPS6KB/p70S6K) and SESN2 levels in the cells with vector or 3×Flag-PTEN-WT or 3×Flag-PTENS113A after TPT treatment. Increased p-JUN-SESN2-p-AMPK and reduced p-RPS6KB expression were observed in the cells with 3×Flag-PTEN-WT compared with the cells with 3×Flag-PTENS113A following TPT treatment (Fig. 7A). The same results were obtained using A549 cells with PTEN shRNA-mediated knockdown (Fig. S6A). JUN or 13
AMPK siRNA respectively inhibited JUN or AMPK activation. A549 cells and HeLa cells with reduced JUN or AMPK expression due to siRNA treatment were exposed to 3 mg/mL TPT for 24 h. As shown in Fig. 7B, the p-JUN, p-AMPK and SESN2 levels were significantly enhanced and p-RPS6KB reduced in the TPT treatment group. However, knockdown of JUN by siRNA significantly reduced the p-JUN, p-AMPK and SESN2 protein levels and p-RPS6KB increased. AMPK activation plays a key role in autophagic induction.
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As shown in Fig. 7C, the p-AMPK levels were significantly enhanced and p-RPS6KB reduced in the TPT
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treatment group. Then, knockdown of AMPK markedly decreased the p-AMPK protein levels and p-RPS6KB increased again. At the same time, Flag and PTEN overexpression was detected in the groups,
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and MAP1LC3-II/LC3-II expression was significantly increased in all TPT-treated groups. A relatively high
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level of MAP1LC3-II/LC3-II was detected in the 3×Flag-PTEN-WT + TPT group in both A549 and HeLa cells (Fig. 7B and C). However, there was no difference in phospho-MAPK(8/9)/JNK(1/2) levels in the
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cells with 3×Flag-PTEN-WT or 3×Flag-PTENS113A following TPT treatment (data not shown). The same results were obtained using A549 cells with PTEN shRNA-mediated knockdown (Fig. S6B and C).
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Collectively, these results suggest that PTEN nuclear translocation promotes autophagy via a mechanism
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associated with p-JUN-SESN2-AMPK pathway activation in A549 and HeLa cells. Moreover, many studies have demonstrated that PTEN is a known inhibitor of AKT, which negatively
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regulates autophagy. We sought to determine whether PTEN phosphorylation affected the AKT pathway.
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A549 cells were transiently transfected with vector and the 3×Flag-PTEN-WT and 3×Flag-PTENS113A plasmids and treated with TPT for an additional 24 h. As shown in Fig. 7D, p-GSK3B and p-AKT levels were not different in the cells with vector or 3×Flag-PTEN-WT or 3×Flag-PTENS113A following TPT treatment. The results suggested that PTEN phosphorylation has no effect on the AKT pathway. PTEN inactivation results in the constitutive activation of the PI3K-AKT pathway and a subsequent increase in protein synthesis, cell cycle progression, migration, and survival.4,5 14
Discussion This study demonstrates for the first time that ATM activation by TPT directly phosphorylates PTEN at Ser113 to induce PTEN nuclear translocation. PTEN nuclear translocation triggers autophagy induction through activation of the p-JUN-SESN2-AMPK pathway in response to TPT treatment.
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ATM, a phospholipid acyl enzyme-3-kinase that directly responds to DNA damage, is mutated in the
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disease ataxia telangiectasia. ATM is activated in response to DNA damage, particularly DNA double-strand breaks (DSBs).34 ATM is involved in various cell signaling events, including the acetylation,
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phosphorylation, and activation of proteins related to cell cycle and DNA damage signaling. In this study,
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we found that ATM kinase was activated by TPT or CDDP treatment in a concentration- and time-dependent manner. Also, we found that endogenous and exogenous PTEN translocates to the nucleus after TPT or
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CDDP treatment, and PTEN translocation is clearly suppressed when ATM activity is inhibited by the ATM specific inhibitor KU55933 or ATM siRNA. These results indicate that DNA damaging agents-induced
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PTEN translocation is dependent of ATM kinase activation. In this study, we first identified a possible ATM phosphorylation site at PTEN serine 113 (Ser113)
pt
using Scansite software. Next, we generated a phospho-specific antibody against PTEN Ser113.
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Coimmunoprecipitation and l-phosphatase were employed to evaluate the specificity of the phospho-PTEN Ser113 antibody. In addition, we found that p-PTEN (Ser113) levels increased in a dose-dependent manner
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in A549 cells and HeLa cells after TPT or CDDP exposure. When ATM expression was reduced by ATM siRNA, TPT-induced PTEN (Ser113) phosphorylation was inhibited. To illustrate the role of PTEN (Ser113) phosphorylation in PTEN nuclear translocation, we transfected the mutated 3×Flag-PTENS113A plasmid and the wild-type 3×Flag-PTEN-WT plasmid into HeLa cells. After treatment with TPT, we found that PTEN did not translocate to the nucleus in the cells harboring the 3×Flag-PTENS113A plasmids. Furthermore，the in vitro phosphorylation assay with purified proteins demonstrated that PTEN was directly phosphorylated by ATM. 15
PTEN activity, localization and stability are regulated by acetylation, ubiquitination, oxidation, and phosphorylation. CSNK2/CK2 (casein kinase 2) primarily phosphorylates PTEN at Ser370 and Ser385, and GSK3B (glycogen synthase kinase 3 beta) phosphorylates PTEN at Ser362 and Thr366.35,36 GLTSCR2/PICT-1 (glioma tumor suppressor candidate region 2) promotes PTEN phosphorylation at Ser380.37 ROCK (RhoA-associated, coiled-coil containing kinase) phosphorylates PTEN at Ser229, Thr232,
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Thr319 and Thr321 in the C2 domain to activate and target PTEN to the plasma membrane.38
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Phospho-PTEN typically refers to PTEN characterized by cytoplasmic localization, enhanced stability, lower activity, and a closed structure. Our findings indicate that Ser113 is a new PTEN phosphorylation site
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that promotes PTEN nuclear translocation. SUMOylation is also a post-translational modification involved
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in various cellular processes. Bassi C et al. report that SUMOylation of PTEN controlled its nuclear localization. They indicate that unlike wild-type SUMO-PTEN, which can rapidly be excluded from the
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nucleus, the SUMO-PTENS/T398A mutant cannot exit the nucleus in cells exposed to ionizing radiation.25 In our study, PTEN-WT and PTENS113A were SUMOylated upon treatment with TPT. However, when we
ed
treated cells with nuclear-export inhibitor Leptomycin B, our results indicated that the PTENS113A mutant
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could enter the nucleus but could not be retained there. It suggested that ATM-mediated PTEN (Ser113) phosphorylation was not involved in PTEN SUMOylation. Our findings indicate that Ser113 is a new PTEN
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phosphorylation site that promotes PTEN nuclear translocation.
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As a tumor suppressor, nuclear PTEN may also maintain normal chromosomal structure and function. Based on the nuclear functions of PTEN, we speculate that ATM-mediated PTEN nuclear translocation affects DNA damage signaling. Using g-H2AFX staining as an indicator of DSBs, we found that TPT-induced DNA damage signaling is perturbed in A549 cells and HeLa cells. Moreover, inhibition of PTEN nuclear translocation increases the number of cells with g-H2AFX foci staining. Our findings reveal that PTEN nuclear translocation, which is regulated by phosphorylation of PTEN at Ser113, is required to 16
repair DNA damage. This finding is consistent with previous studies. Moreover, DNA break-repair is affected by the cell cycle stage. TPT-stimulated apoptosis has been reported in S-phase A549 cells.28 In this study, we found that PTENS113A mutation had no effect on cell cycle stage and cell apoptosis. Autophagy is characterized by the sequestration and delivery of bulk cytoplasm and organelles in double or multimembrane autophagic vesicles for subsequent degradation by the cell’s own lysosomal
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system.39 During the formation of autophagosomes, MAP1LC3/LC3 is lipidated. The subsequent
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LC3-phospholipid conjugate (MAP1LC3-II/LC3-II) is localized to autophagosomes and autolysosomes, and MAP1LC3-II/LC3-II accumulation is used as a marker of autophagy.29 Furthermore, after PTEN nuclear
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translocation, levels of cytoplasmic PTEN are hypothesized to decrease, leading to PI3K-AKT pathway
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activation and sequential inhibition of autophagic induction. In contrast, we found that autophagic induction significantly increased in the cells harboring the 3×Flag-PTEN-WT plasmid compared with the
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3×Flag-PTENS113A plasmid. This indicated that PTEN nuclear translocation promotes autophagic induction following TPT treatment.
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How does PTEN nuclear translocation contribute to autophagic induction? SESN2 is a TP53 target gene
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that activates the AMPK pathway to induce autophagy. However, TP53 is mutated and nonfunctional in A549 and HeLa cells; thus, TP53 is not involved in the regulation of SESN2 expression in these 2 cell lines.
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We have previously demonstrated that activated JUN activates SESN2 expression at the transcriptional level
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through binding to the promoter of SESN2, potentially activating AMPK to induce autophagy through inhibition of MTORC1 (mechanistic target of rapamycin complex 1) in colon cancer cells.31 Here, we found that SESN2 and p-AMPK expression increased and p-RPS6KB expression reduced in the cells with 3×Flag-PTEN-WT following TPT treatment. JUN is a member of the Jun family that consists of JUN, JUNB, and JUND. JUN is a component of the transcription factor activator protein-1 (AP-1).40 In addition, Dunn et al. report that JUN is a principal target of MAPK(8/9)/JNK(1/2). In this study, p-JUN (Ser63), p-AMPK and 17
SESN2 protein levels were relatively increased and p-RPS6KB reduced in the cells harboring 3×Flag-PTEN-WT compared with the cells harboring 3×Flag-PTENS113A after TPT treatment. A relatively high level of MAP1LC3-II/LC3-II was detected in the 3×Flag-PTEN-WT + TPT group in both A549 and HeLa cells. However, p-MAPK(8/9)/JNK(1/2) expression did not vary between the 3×Flag-PTEN-WT + TPT and 3×Flag-PTENS113A + TPT groups (data not show). Therefore, we speculate that PTEN nuclear
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translocation regulates JUN phosphorylation and stability. The level of JUN phosphorylation at the
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C-terminal sites Thr239 and Ser243 has been reported to participate in its downregulation.41 Thus, whether nuclear PTEN affects JUN (Ser243) phosphorylation via its phosphatase activity is of interest.
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In conclusion, the results presented here provide evidence that ATM regulates PTEN phosphorylation at
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Ser113, as a result of exposure to DNA-damaging agents, and promotes PTEN nuclear translocation, which further promotes autophagy through activation of the p-JUN-SESN2-AMPK pathway in tumor cells. We
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conclude that autophagy requires PTEN nuclear translocation that is regulated by PTEN (Ser113)
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Cell culture
pt
Materials and Methods
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phosphorylation in response to DNA-damaging agent treatment in cancer cells.
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The human cervical cancer (HeLa) and lung adenocarcinoma (A549) cell lines were obtained from American Type Culture Collection (CCL-2 and CRM-CCL-185, respectively) and have been stored in liquid nitrogen at our lab. Both cell lines were cultivated in DMEM and RMPI 1640 medium, respectively, supplemented with 10% fetal bovine serum, penicillin (100 μg/mL) and streptomycin (100 μg/mL) in a 5% CO2 humidified atmosphere at 37 ºC. All experiments were performed in these cells between passage 10 and 20 at the confluency of 70%. 18
Drugs and reagents Topotecan (TPT; 004) was purchased from GlaxoSmithKline. KU55933 (587871-26-9) was purchased from Millipore. RPMI-1640 medium (31800-022), DMEM medium (12800-017), fetal bovine serum, the anti-flag antibody, anti-TUBA4A (t6199) and ATP (A1582) were obtained from Sigma Aldrich.
(5883),
anti-HIST1H3A/histone
H3
(9715),
anti-RPS6KB/p70S6K
(2708),
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phospho-ATM
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Anti-SQSTM1/p62 (8025), phospho-H2AFX (γ-H2AFX; 9718), anti-PTEN (9559), anti-ATM (2873),
phospho-RPS6KB/p70S6K (9234) and anti-normal mouse IgG (7076) were purchased from Cell Signaling
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Technology. The anti-MAP1LC3/LC3 antibody (NB100-2220) was obtained from Novus Biologicals.
an
Anti-phospho-PTEN antibody was prepared by the Abgent, Inc. Lipofectamine 2000 (11668-027), goat anti-rabbit Alexa 488 (997773) and opti-MEM medium (22600050) were purchased from the InvitrogenTM.
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Purified ATM protein (active) (14-933) was purchased from Millipore, and purified PTEN recombinant protein (TP710023) was obtained from Origene. ATM siRNA, JUN siRNA and AMPK siRNA were obtained
ed
from GenePharma Co, Ltd. The 3×Flag-PTEN-WT plasmid and the 3×Flag-PTENS113A plasmid were
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(S1726).
pt
prepared by Fulen Gene Company. Leptomycin B was purchased from Beyotime Institute of Biotechnology
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Immunoblotting analysis
A549 and HeLa cells were harvested and lysed in 100 μL of lysis buffer (20 mM Tris-HCl, pH 7.5, 150
mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100 (Sigma-Aldrich, 234729), 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin) after treatment with TPT for the indicated times. Lysates were centrifuged at 13,400 g for 15 min at 4 ºC. The supernatant fraction was then collected. Protein concentrations were determined using the Pierce BCA protein assay kit (Thermo, 19
1862634). Whole cell lysates were prepared by adding 6×SDS-PAGE buffer (Serva, 151-21-3) and incubation at 100 ºC for 10 min for protein denaturation. Equivalent aliquots of protein samples (20 to 40 μg) were loaded and electrophoresed on SDS-PAGE gels and then transferred to polyvinylidene fluoride/PVDF membranes. The membranes were first blocked with 5% nonfat dry milk for 60 min at room temperature and then incubated with primary antibodies overnight at 4 ºC. Next, the blots were incubated with horseradish
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peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, sc-2004 and sc-2005) for 1 h at room
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temperature. After 3 washes with TBST (10 mM Tris, 150 mM NaCl, pH 8.0, 0.1% Tween 20 [MP Biomedicals, 103168]) for 5 min, the proteins were visualized with the ECL chemiluminescence reagent and
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exposure to radiograph film.
Immunofluorescence
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A549 and HeLa cells were seeded in 24-well plates containing NUNC Thermanox™ coverslips. Upon treatment, cells were fixed with a 4% paraformaldehyde fixative solution at room temperature for 20 min
ed
and washed 3 times in 1×PBS (0.8% NaCl, 0.02% KCl, 0.144% Na2HPO4, 0.024% KH2PO4, pH7.4) for 5
pt
min each. Cells were then permeabilized with 0.25% Octoxynol 9/Triton X-100 (Sigma-Aldrich, 9002-93-1) in PBS at 4 °C for 10 min and blocked with 4% BSA (MP Biomedicals, 0218054991) blocking solution at
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37 ºC for 30 min. The cells were incubated with the primary antibody (1:300) overnight at 4 ºC. Cells were
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washed thrice in PBST for 5 min and incubated with the fluorescent secondary antibody at room temperature for 1 h. DAPI was added for 5 min to visualize nuclei. After using an antifluorescence quenching reagent, the coverslips were stored at 4 ºC. Cells were observed using a laser scanning confocal microscope (Olympus, FV-1000).
shRNA and siRNA transfection The target sequence for ATM or JUN or AMPK siRNA was respectively 5'-AAGGTGAA 20
GATATATTCCTCC-3',5'-UGCCUACCAUCUCAUAAUAtt-3', 5′-AGAUGG AAACGACCUUCUATT-3′. The hairpin RNA (shRNA) sequence targeting PTEN was 5'-GATCTTGACCAATGGCTAAGT-3'. The control siRNA sequence was 5'-UUC UCC GAA CGU GUC ACG UTT-3' and synthesized by GenChem Co. HeLa and A549 cells were seeded into 6-well plates the day before transfection. When the cells reached 40% to 60% confluence, transfection was performed using serum-free Opti-MEM media (Gibco, 31985-070), lipofectamine 2000 (Invitrogen, 11668-019) and siRNA according to the manufacturer’s recommendations.
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The medium was replaced with fresh growth medium containing 10% fetal bovine serum 6 h after
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transfection, and cells were incubated for an additional 24 h for subsequent experiments.
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In vitro phosphorylation assay
This assay was carried out using commercially available active ATM kinase (1 μg) and PTEN
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recombinant protein (1 μg). Reaction was initiated by adding ATP and carried out at 30 °C in 25 μL kinase buffer [20 mM Tris·HCl (pH 7.5), 2 mM EDTA, 10 mM MgCl2, 1 mM DTT] for 30 min. The reaction was
Cell cycle analysis
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terminated by adding 12.5 μL 3x loading buffer and incubating at 100 ºC for 10 min.
pt
Cell cycle analysis was carried out by flow cytometry. Briefly, A549 cells were seeded into 6-well culture plates, treated as indicated, collected, fixed, stained with propidium iodide (1 μg/mL) and flow
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cytometric analysis on a FACScan with a 488 nm Argon laser (Backman Coulter, FC500). Quantitation was
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performed by scoring cells for sub-G0/G1 DNA content using the SigmaPlot software.
In vitro SUMOylation HeLa cells were transiently transfected with 10 μg of the HA-SUMO1 and 10 μg 3×Flag-PTEN-WT or 3×Flag-PTENS113A plasmid. Equal amounts of protein sample were incubated with a flag monoclonal antibody for 2 h at 4 ºC. The antibody-protein complex was mixed with protein A Sepharose 4 fast flow 21
solution for 16 h at 4 ºC. After centrifugation, the pellet fraction was washed 4 times and resuspended in loading buffer. The suspension was boiled for 10 min at 100 ºC, centrifuged and analyzed by SDS–PAGE and western blotting with anti-HA antibody.
Plasmids and transfection
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The vector pcDNA3.1 and YFP-LC3 plasmids were kindly gifted by Dr. Rong Deng, Dr. Dandan Li
cr
and Dr. Xiaoqi Wu, Cancer Center, Sun Yat-sen University, Guangzhou, China. The 3×Flag-PTEN-WT and 3×Flag-PTENS113A, 3×Flag-PTEN-WT (shPTEN-resistant) and 3×Flag-PTENS113A (shPTEN-resistant)
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plasmids were produced by the Gene Copoeia Company. HA-SUMO1 plasmid was purchased from
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Addgene (plasmid 21154). All mutations were verified by DNA sequencing. Transfection was performed as
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previously described.42,43
Cytoplasmic and nuclear protein extraction
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Cytosolic and nuclear fractions were prepared using a nuclear-cytosolic protein isolation kit (Beyotime Institute of Biotechnology). HeLa and A549 cells were seeded in 10 cm plates. After treatment with 3
pt
mg/mL TPT for 24 h, cells were collected and washed with 1 mL of PBS and then centrifuged in 4 ºC at 90 g
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for 3 min. The resultant supernatant fraction was discarded, and the sediment was resuspended in reagent A
Ac
with fresh PMSF. Samples were vortexed vigorously for 5 sec and placed on ice for 15 min before adding reagent B. The samples were centrifuged at 13,400 g for 3 min at 4 ºC. The supernatant fraction containing the cytosolic proteins was collected and used immediately or stored at -80 ºC. The sediment containing the nuclear proteins was resuspended in nuclear protein extraction reagent with PMSF. The samples were vortexed at 13,400 g for 30 sec, placed on ice for 2 min, and vortexed for 30 sec. The last 2 steps were repeated for 30 min, and the samples were centrifuged at 13,400 g for 10 min at 4 ºC. The supernatant
22
fractions containing the nuclear proteins was used immediately or stored at -80 ºC. The concentrations of the cytosolic and nuclear proteins were measured using the bicinchoninic acid/BCA method (Thermo, 1862634).
Coimmunoprecipitation
ip t
A549 and HeLa cells were transiently transfected with the 3×Flag-PTEN-WT plasmid. Cells were lysed
cr
with RIPA lysis buffer, and the protein concentration for each group was measured using the bicinchoninic acid method. Equal amounts of protein sample were incubated with a flag monoclonal antibody for 2 h at 4
us
ºC. The antibody-protein complex was mixed with Protein A/G Agarose (Roche, 11243233001) for 16 h at 4
an
ºC. After centrifugation, the pellet fraction was washed 4 times and resuspended in loading buffer. The suspension was boiled for 10 min at 100 ºC and centrifuged, and the supernatant fraction was used for
Statistical analysis
ed
M
western blotting to identify the coprecipitated proteins.
pt
All assays were performed in triplicate. Data are expressed as the mean ± standard deviation (SD). Statistical analyses were performed with the 2-tailed Student t test for comparison of 2 groups by SPSS 13.0
Ac
ce
software. P
