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Assay of the redox state of the tumor suppressor PTEN by mobility shift
Methods 77–78 (2015) 58–62
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Methods journal homepage: www.elsevier.com/locate/ymeth
Assay of the redox state of the tumor suppressor PTEN by mobility shift Seong-Jeong Han a,b, Younghee Ahn c, Iha Park a, Ying Zhang a, Inyoung Kim a, Hyun Woo Kim a, Chang-Sub Ku d, Kee-Oh Chay a, Sung Yeul Yang a, Bong Whan Ahn a, Dong Il Jang d, Seung-Rock Lee a,⇑ a Department of Biochemistry, Department of Biomedical Science, Research Center for Aging and Geriatrics, Research Institute of Medical Sciences, Chonnam National University Medical School, Gwangju 501-190, Republic of Korea b School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea c Department of Pediatrics and Clinical Neuroscience, Alberta Children’s Hospital Research Institute for Child and Maternal Health, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N4N1, Canada d COTDE Inc. 19-3, Ugakgol-gil, Susin-myeon, Cheonan-si, Chungcheongnam-do 330-882, Republic of Korea
a r t i c l e
i n f o
Article history: Received 22 September 2014 Received in revised form 14 January 2015 Accepted 19 January 2015 Available online 27 January 2015 Keywords: PTEN Redox regulation Mobility shift assay N-Ethylmaleimide
a b s t r a c t PTEN is reversibly oxidized in various cells by exogenous hydrogen peroxide as well as by endogenous hydrogen peroxide generated when cells are stimulated with growth factors, cytokines and hormones. A gel mobility shift assay showed that oxidized PTEN migrated more rapidly than reduced PTEN on a non-reducing SDS–PAGE gel. Oxidized PTEN was reduced when treated with dithiothreitol. Supplementation of N-ethylmaleimide in the cell lysis buffer was critical for the apparent bands of oxidized and reduced PTEN. Formation of oxidized PTEN was abolished when the active site Cys124 or nearby Cys71 was replaced with Ser suggesting that Cys124 and Cys71 are involved in the formation of an intramolecular disulfide bond. These results show that the mobility shift assay is a convenient method to analyze the redox state of PTEN in cells. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), are well known to be important second messengers in intracellular redox signaling and interact directly with various signaling molecules to regulate physiological processes such as inflammation, cell proliferation, survival and apoptosis, and angiogenesis and metastasis [1]. Thiols and selenols are oxidized by ROS and play an important role in maintaining cellular reduction/ oxidation (redox) homeostasis. The reactivity of cysteine and selenocysteine residues plays a key role in redox sensors and switches and affects the catalytic and metal binding capacity of proteins [2–4]. Members of the PTPs family possess a unique cysteine residue, which is highly conserved in the signature active site motif, HisCys-X-X-Gly-X-X-Arg-Ser/Thr (where X is any amino acid), and has a low pKa (4.7–5.4), which exists as a thiolate anion at neutral pH. The thiolate anion of cysteine residue in the active site is very susceptible to oxidation by H2O2 [5]. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a member of the protein tyrosine phosphate family. PTEN acts to negatively regulate
⇑ Corresponding author. Fax: +82 62 223 8321. E-mail address: [email protected] (S.-R. Lee). http://dx.doi.org/10.1016/j.ymeth.2015.01.007 1046-2023/Ó 2015 Elsevier Inc. All rights reserved.
the PI3K/AKT pathway by catalyzing dephosphorylation of the phosphate attached to the 30 -hydroxyl group of phosphatidylinositol (3,4,5)-triphosphate (PIP3) [6,7]. PTEN is reversibly oxidized in various cells such as in macrophages when treated with phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS) [7]. Reversible oxidation of PTEN has also been observed in neuroblastoma cells stimulated with insulin using gel mobility shift assay [8]. PTEN oxidation has been observed in HEK293 cells stimulated with insulin, HeLa cells stimulated with EGF and NIH3T3 fibroblasts stimulated with PDGF using mobility shift assays in addition to biotinylation and alkylation protection assay methods [8,9]. Oxidized PTEN is also detected using gel mobility shift assay when cells are exposed to reactive oxygen and nitrogen species [10,11] and arsenic trioxide (As2O3) [12]. PTEN oxidation is implicated in tumorigenesis [13], angiogenesis [14], acquired long QT syndrome [12] and aging [15]. PTEN oxidation is inhibited by peroxiredoxin II (Prx II), which removes H2O2 generated in response to growth factors [9,13]. Oxidized PTEN is reduced by intracellular reducing systems such as glutathione (GSH) and thioredoxin (Trx) in vitro [16,17]. N-ethylmaleimide (NEM) is a well-known alkylating reagent, and it is used to limit the occurrence of various reactions of thiols and selenols. NEM is a more effective reagent for alkylating thiols than iodoacetamide (IAM) and iodoacetic acid (IAA) because it
S.-J. Han et al. / Methods 77–78 (2015) 58–62
functions over a wide pH range [18]. In this report, we describe the gel mobility shift assay for assessing the redox state of PTEN using NEM as an alkylating reagent. 2. Supplementation of N-ethylmaleimide 2.1. Principle Cellular thiols and selenols are susceptible to redox reactions. Generally, N-ethylmaleimide (NEM) is a more sensitive and effective alkylation reagent than iodoacetic acid (IAA) and iodoacetamide (IAM) for blocking redox reactions of cellular thiols and selenols. Thus, we investigated whether supplementation of NEM into cell lysis buffer affects the assay of the redox state of the tumor suppressor PTEN in cells. 2.2. Procedure 2.2.1. Preparation of a PTEN-specific antibody A polyclonal antiserum was raised in rabbits. The human PTEN sequence (GenBank Accession No. NP000305) was analyzed to identify antigen epitopes. Rabbits were immunized simultaneously with KLH conjugated to two synthetic peptides containing the deduced amino acids Ac-8IVSRNKRRYQEDGFD22-C and C-388ENEPFDEDQHTQI400-NH2, The ends of the synthetic peptides were appended with a cysteine or blocked by amidation or acetylation at each ends to better mimic an internal amino acid sequence. Synthetic peptides and affinity gel matrix columns were manufactured by Peptron (Daejeon, Korea). Rabbits were boosted three times every two weeks. To confirm the immune response by Western blotting and ELISA analysis, the rabbit was sacrificed and serum was collected by centrifugation. Affinity gel matrix columns were washed with 10 volumes of phosphate-buffered saline (PBS) and equilibrated with 5 volumes of 10 mM Tris buffer (pH 7.5). Before loading the antiserum, it was mixed with the same volume of 10 mM Tris buffer (pH 7.5). Purified anti-PTEN was eluted with 100 mM glycine (pH 2.5), and the antibody fractions were neutralized in 1 M Tris–HCl (pH 8.0) buffer. The sample was resolved on a SDS–PAGE gels and visualized by Coomassie brilliant blue staining or Western blot analysis. 2.2.2. Cell culture and preparation of samples HeLa cells and NIH3T3 cells were maintained with Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS; Hyclone, USA). All cells were cultured in a humidified air containing 5% CO2 at 37 °C, and grown to 80% confluency. Then, cells were washed with PBS and changed to serum-free medium. Next, the cells were treated with hydrogen peroxide (H2O2) at the various concentrations and times indicated. After incubation for the indicated times, 1 lg/mL catalase was added and incubated for 5 min. Cells were washed with PBS. Next, 200 lL of lysis buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5% glycerol, 0.1% NP40, 100 lM PMSF, with or without NEM, IAM and IAA) was added into each well of the cultured cells. NEM was solubilized in dimethylformamide (Sigma, USA) immediately before the use. Cell lysates were sonicated and centrifuged (15,000 rpm, 10 min). Supernatants were transferred to new tubes, and protein concentrations were measured with a BCA protein assay kit (Pierce, USA). 2.2.3. Non-reducing/reducing SDS–PAGE and Western blot analysis Cell lysates samples were mixed in 5 SDS sample buffer (60 mM Tris, pH 6.8, 25% glycerol, 2% SDS, 0.05% bromophenol) and then boiled at 95 °C for 5 min. Non-reducing samples were prepared without the use of thiol reducing agents, such as dithio-
59
threitol (DTT) and 2-mercaptoethaonl. Samples were separated on 8% SDS–PAGE gels, and the separated proteins were transferred to nitrocellulose membranes. To reduce non-specific binding, the membranes were incubated with 5% skim milk for 1 h at room temperature. Then, primary PTEN antibodies were incubated for 1.5 h at room temperature. The membranes were washed with TBST (25 mM Tris, pH 7.4, 150 mM NaCl, 2 mM KCl, and 0.01% Tween 20), and incubated with anti-rabbit IgG horseradish peroxidase-conjugated antibody (Ab Frontier, Daejeon, Korea) for 1 h at room temperature, and then re-washed with TBST buffer. Immunoreactive proteins were visualized using an enhanced chemiluminescence detection system (Pierce, USA). The experimental procedure is shown in Fig. 1.
2.2.4. Plasmids and transient transfection The plasmid for the N-terminal HA-tagged wild-type or PTEN mutants were cloned into the pCGN vector [10]. NIH3T3 cells were transiently transfected with plasmids using the PolyFect reagent (Qiagen) according to the manufacturer’s protocol or using the calcium phosphate precipitation method. At 24 h after transfection, cells were washed and treated with hydrogen peroxide (H2O2) for the times indicated.
3. Results Hydrogen peroxide is a well-known and commonly used as an oxidant to induce oxidation and inactivation of PTEN. HeLa cells were incubated for 5 min with 1 mM H2O2. Cells were lysed with lysis buffer supplemented with various concentrations of NEM, IAM and IAA, and then subjected to immunoblot analysis using non-reducing SDS–PAGE gels. As a result, samples supplemented with more than 1 mM NEM in the lysis buffer showed apparent bands of oxidized and reduced PTEN (Fig. 2A). In contrast, the bands of oxidized and reduced PTEN were not clear and blurred in the samples supplemented with lower than 1 mM NEM. Samples with 1 mM IAM and 10 mM IAM showed apparent bands of oxidized and reduced PTEN on the non-reducing SDS–PAGE gel. However, samples with 10 mM IAM showed no bands of PTEN on the reducing SDS–PAGE gel because 10 mM IAM caused the aggregation and precipitation of proteins immediately after mixing samples with reducing SDS–PAGE sample buffer (Fig. 2B). The bands of PTEN were not apparent in the samples supplemented with IAA (Fig. 2C).
Fig. 1. The scheme of experimental procedure for the assay of the redox state of PTEN by mobility shift.
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Fig. 2. Effect of NEM, IAM and IAA on the mobility of oxidized and reduced PTEN in HeLa cells. Cells were incubated in the absence or presence of 1 mM H2O2 for 5 min and then lysed with lysis buffer supplemented with various concentrations of NEM (A), IAM (B) and IAA (C) as indicated. Cell lysates (20 lg total protein) were resolved by nonreducing and reducing SDS–PAGE and subjected to Western blotting analysis with anti-PTEN and b-actin antibodies. b-actin levels were used as a loading control.
Next, we examined that oxidation of PTEN by H2O2 in cells at various concentrations and incubated for different times, after which cells were lysed with or without 10 mM NEM-supplemented lysis buffer and then subjected to immunoblot analysis with PTEN antibodies. Oxidized and reduced PTEN were apparently detected in the samples supplemented with 10 mM. The oxidized form of PTEN was increased in a dose-dependent manner and then declined. In contrast, in the samples without NEM, we did not identify oxidized PTEN bands (Fig. 3). These data suggested that supplementation of NEM in the lysis buffer and separation of lysate samples by non-reducing SDS–PAGE is crucial and effective to assess the redox state of PTEN. Previously, we showed that formation of oxidized PTEN band was abolished when Cys124 or Cys71 residues were replaced by Ser residue using recombinant wild type and mutant PTEN protein in vitro [10]. To examine the effects of mutation of Cys71 and Cys124 residues on the mobility shift of PTEN in cells, wild-type and mutant PTEN were expressed in NIH3T3 cells, which were treated with 0.7 mM H2O2 and incubated for the indicated times. Then, cell lysates were alkylated with 10 mM NEM and subjected to non-reducing SDS–PAGE and Western blotting with HA-specific antibodies. As a result, the oxidized PTEN band was not detected in the samples expressing Cys71 and Cys124 single mutant and Cys71/ Cys124 double mutant (Fig. 4). These results suggest that the
Cys71 and Cys124 are involved in the formation of an intramolecular disulfide bond that result in rapid migration of oxidized PTEN.
4. Concluding remarks PTEN is a well-known tumor suppressor. It is oxidized by ROS with the formation of an intracellular disulfide between the active site Cys124 and nearby Cys71 residues. PTEN oxidation inactivates the catalytic activity, causing elevation of AKT activity and downstream cascade signaling, resulting in deregulation of cell growth and proliferation. Oxidized PTEN is reversibly reduced over time after oxidative stress by H2O2. Cellular redox homeostasis is controlled by thioredoxin (Trx) and/or glutaredoxin (Grx) [17,19,20] Thus, changes in intracellular levels of these molecules can influence the redox status of PTEN. Therefore, redox regulation of PTEN play a key roles in tumorigenesis. Alkylating reagents are used to halt in the thiol–disulfide status in cells. N-ethylmaleimide (NEM), iodoacetamide (IAM), and iodoacetic acid (IAA) are commonly used for protein thiol alkylation. However, there is distinct difference in experimental approaches. IAA and IAM react irreversibly with thiols in nucleophilic substitution reactions to form the carboxymethyl or carboxamidomethyl derivatives. It is light sensitive and must be protected from lights during reactions. Moreover, to complete alkylation of protein thi-
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Fig. 3. Assay of redox state of PTEN with lysis buffer supplemented with NEM. Cells were treated with 1 mM H2O2 for the times indicated (0, 5, 10, 30, 60, 120 min) (A) and with various concentrations (0, 0.1, 0.5, 1, 2, 4 mM) for 10 min (B). Cells were lysed with lysis buffer supplemented with 10 mM NEM (right) or without NEM (left) and subjected to non-reducing and reducing SDS–PAGE followed by Western blot analysis.
Fig. 4. Effects of mutation of Cys71 or Cys124 residues on the mobility of PTEN. NIH3T3 cells expressing HA-tagged wild-type PTEN or C121S, C74S, C121/74S mutants were treated with 0.7 mM H2O2 for the times indicated (0, 2, 5, 10, 30, 90 min). Cells were lysed with lysis buffer supplemented with 10 mM NEM. Then, samples were subjected to non-reducing SDS–PAGE followed by Western blot analysis with a mouse anti-HA antibody.
ols, it is required for higher concentrations, pHs and reaction times. In contrast, the reaction of NEM with thiols is Michael-type addition reaction which across the double bond to form a thioether bond between the thiol and the maleimide. When compared with other alkylation reagent, the reaction of thiol alkylation with NEM requires less time and less dependent on pHs. Thus, NEM is more effective alkylation reagents than IAA, IAM and uncharged NEM can react in hydrophobic conditions [18,21]. In the previous studies for monitoring of intracellular thiols status of protein, cysteine residues are conjugated with alkylating reagents such as NEM, IAA and IAM that were biotin-tagged, fluorescently active, or radioactive [22–24]. Biotin labeling methods were subsequently measured by immunoblotting with streptavidin conjugated-HRP antibody. However, these methods are complicated and depend on reagent properties and on yield of reactions. In contrast, the gel mobility shift assay described in this report required only suitable antibody and NEM. In conclusion, the mobility shift assay using NEM as an alkylating agent, non-reduc-
ing SDS–PAGE and Western blotting with specific antibody was an effective and convenient method to assess redox state of PTEN in cells. This method might contribute to accelerate studies for identifying proteins involved in the redox regulation of PTEN in cells and disease models.
Acknowledgments This study was supported by a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No. HN11C0047).
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Contents lists available at ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Assay of the redox state of the tumor suppressor PTEN by mobility shift Seong-Jeong Han a,b, Younghee Ahn c, Iha Park a, Ying Zhang a, Inyoung Kim a, Hyun Woo Kim a, Chang-Sub Ku d, Kee-Oh Chay a, Sung Yeul Yang a, Bong Whan Ahn a, Dong Il Jang d, Seung-Rock Lee a,⇑ a Department of Biochemistry, Department of Biomedical Science, Research Center for Aging and Geriatrics, Research Institute of Medical Sciences, Chonnam National University Medical School, Gwangju 501-190, Republic of Korea b School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea c Department of Pediatrics and Clinical Neuroscience, Alberta Children’s Hospital Research Institute for Child and Maternal Health, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N4N1, Canada d COTDE Inc. 19-3, Ugakgol-gil, Susin-myeon, Cheonan-si, Chungcheongnam-do 330-882, Republic of Korea
a r t i c l e
i n f o
Article history: Received 22 September 2014 Received in revised form 14 January 2015 Accepted 19 January 2015 Available online 27 January 2015 Keywords: PTEN Redox regulation Mobility shift assay N-Ethylmaleimide
a b s t r a c t PTEN is reversibly oxidized in various cells by exogenous hydrogen peroxide as well as by endogenous hydrogen peroxide generated when cells are stimulated with growth factors, cytokines and hormones. A gel mobility shift assay showed that oxidized PTEN migrated more rapidly than reduced PTEN on a non-reducing SDS–PAGE gel. Oxidized PTEN was reduced when treated with dithiothreitol. Supplementation of N-ethylmaleimide in the cell lysis buffer was critical for the apparent bands of oxidized and reduced PTEN. Formation of oxidized PTEN was abolished when the active site Cys124 or nearby Cys71 was replaced with Ser suggesting that Cys124 and Cys71 are involved in the formation of an intramolecular disulfide bond. These results show that the mobility shift assay is a convenient method to analyze the redox state of PTEN in cells. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), are well known to be important second messengers in intracellular redox signaling and interact directly with various signaling molecules to regulate physiological processes such as inflammation, cell proliferation, survival and apoptosis, and angiogenesis and metastasis [1]. Thiols and selenols are oxidized by ROS and play an important role in maintaining cellular reduction/ oxidation (redox) homeostasis. The reactivity of cysteine and selenocysteine residues plays a key role in redox sensors and switches and affects the catalytic and metal binding capacity of proteins [2–4]. Members of the PTPs family possess a unique cysteine residue, which is highly conserved in the signature active site motif, HisCys-X-X-Gly-X-X-Arg-Ser/Thr (where X is any amino acid), and has a low pKa (4.7–5.4), which exists as a thiolate anion at neutral pH. The thiolate anion of cysteine residue in the active site is very susceptible to oxidation by H2O2 [5]. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a member of the protein tyrosine phosphate family. PTEN acts to negatively regulate
⇑ Corresponding author. Fax: +82 62 223 8321. E-mail address: [email protected] (S.-R. Lee). http://dx.doi.org/10.1016/j.ymeth.2015.01.007 1046-2023/Ó 2015 Elsevier Inc. All rights reserved.
the PI3K/AKT pathway by catalyzing dephosphorylation of the phosphate attached to the 30 -hydroxyl group of phosphatidylinositol (3,4,5)-triphosphate (PIP3) [6,7]. PTEN is reversibly oxidized in various cells such as in macrophages when treated with phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS) [7]. Reversible oxidation of PTEN has also been observed in neuroblastoma cells stimulated with insulin using gel mobility shift assay [8]. PTEN oxidation has been observed in HEK293 cells stimulated with insulin, HeLa cells stimulated with EGF and NIH3T3 fibroblasts stimulated with PDGF using mobility shift assays in addition to biotinylation and alkylation protection assay methods [8,9]. Oxidized PTEN is also detected using gel mobility shift assay when cells are exposed to reactive oxygen and nitrogen species [10,11] and arsenic trioxide (As2O3) [12]. PTEN oxidation is implicated in tumorigenesis [13], angiogenesis [14], acquired long QT syndrome [12] and aging [15]. PTEN oxidation is inhibited by peroxiredoxin II (Prx II), which removes H2O2 generated in response to growth factors [9,13]. Oxidized PTEN is reduced by intracellular reducing systems such as glutathione (GSH) and thioredoxin (Trx) in vitro [16,17]. N-ethylmaleimide (NEM) is a well-known alkylating reagent, and it is used to limit the occurrence of various reactions of thiols and selenols. NEM is a more effective reagent for alkylating thiols than iodoacetamide (IAM) and iodoacetic acid (IAA) because it
S.-J. Han et al. / Methods 77–78 (2015) 58–62
functions over a wide pH range [18]. In this report, we describe the gel mobility shift assay for assessing the redox state of PTEN using NEM as an alkylating reagent. 2. Supplementation of N-ethylmaleimide 2.1. Principle Cellular thiols and selenols are susceptible to redox reactions. Generally, N-ethylmaleimide (NEM) is a more sensitive and effective alkylation reagent than iodoacetic acid (IAA) and iodoacetamide (IAM) for blocking redox reactions of cellular thiols and selenols. Thus, we investigated whether supplementation of NEM into cell lysis buffer affects the assay of the redox state of the tumor suppressor PTEN in cells. 2.2. Procedure 2.2.1. Preparation of a PTEN-specific antibody A polyclonal antiserum was raised in rabbits. The human PTEN sequence (GenBank Accession No. NP000305) was analyzed to identify antigen epitopes. Rabbits were immunized simultaneously with KLH conjugated to two synthetic peptides containing the deduced amino acids Ac-8IVSRNKRRYQEDGFD22-C and C-388ENEPFDEDQHTQI400-NH2, The ends of the synthetic peptides were appended with a cysteine or blocked by amidation or acetylation at each ends to better mimic an internal amino acid sequence. Synthetic peptides and affinity gel matrix columns were manufactured by Peptron (Daejeon, Korea). Rabbits were boosted three times every two weeks. To confirm the immune response by Western blotting and ELISA analysis, the rabbit was sacrificed and serum was collected by centrifugation. Affinity gel matrix columns were washed with 10 volumes of phosphate-buffered saline (PBS) and equilibrated with 5 volumes of 10 mM Tris buffer (pH 7.5). Before loading the antiserum, it was mixed with the same volume of 10 mM Tris buffer (pH 7.5). Purified anti-PTEN was eluted with 100 mM glycine (pH 2.5), and the antibody fractions were neutralized in 1 M Tris–HCl (pH 8.0) buffer. The sample was resolved on a SDS–PAGE gels and visualized by Coomassie brilliant blue staining or Western blot analysis. 2.2.2. Cell culture and preparation of samples HeLa cells and NIH3T3 cells were maintained with Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS; Hyclone, USA). All cells were cultured in a humidified air containing 5% CO2 at 37 °C, and grown to 80% confluency. Then, cells were washed with PBS and changed to serum-free medium. Next, the cells were treated with hydrogen peroxide (H2O2) at the various concentrations and times indicated. After incubation for the indicated times, 1 lg/mL catalase was added and incubated for 5 min. Cells were washed with PBS. Next, 200 lL of lysis buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5% glycerol, 0.1% NP40, 100 lM PMSF, with or without NEM, IAM and IAA) was added into each well of the cultured cells. NEM was solubilized in dimethylformamide (Sigma, USA) immediately before the use. Cell lysates were sonicated and centrifuged (15,000 rpm, 10 min). Supernatants were transferred to new tubes, and protein concentrations were measured with a BCA protein assay kit (Pierce, USA). 2.2.3. Non-reducing/reducing SDS–PAGE and Western blot analysis Cell lysates samples were mixed in 5 SDS sample buffer (60 mM Tris, pH 6.8, 25% glycerol, 2% SDS, 0.05% bromophenol) and then boiled at 95 °C for 5 min. Non-reducing samples were prepared without the use of thiol reducing agents, such as dithio-
59
threitol (DTT) and 2-mercaptoethaonl. Samples were separated on 8% SDS–PAGE gels, and the separated proteins were transferred to nitrocellulose membranes. To reduce non-specific binding, the membranes were incubated with 5% skim milk for 1 h at room temperature. Then, primary PTEN antibodies were incubated for 1.5 h at room temperature. The membranes were washed with TBST (25 mM Tris, pH 7.4, 150 mM NaCl, 2 mM KCl, and 0.01% Tween 20), and incubated with anti-rabbit IgG horseradish peroxidase-conjugated antibody (Ab Frontier, Daejeon, Korea) for 1 h at room temperature, and then re-washed with TBST buffer. Immunoreactive proteins were visualized using an enhanced chemiluminescence detection system (Pierce, USA). The experimental procedure is shown in Fig. 1.
2.2.4. Plasmids and transient transfection The plasmid for the N-terminal HA-tagged wild-type or PTEN mutants were cloned into the pCGN vector [10]. NIH3T3 cells were transiently transfected with plasmids using the PolyFect reagent (Qiagen) according to the manufacturer’s protocol or using the calcium phosphate precipitation method. At 24 h after transfection, cells were washed and treated with hydrogen peroxide (H2O2) for the times indicated.
3. Results Hydrogen peroxide is a well-known and commonly used as an oxidant to induce oxidation and inactivation of PTEN. HeLa cells were incubated for 5 min with 1 mM H2O2. Cells were lysed with lysis buffer supplemented with various concentrations of NEM, IAM and IAA, and then subjected to immunoblot analysis using non-reducing SDS–PAGE gels. As a result, samples supplemented with more than 1 mM NEM in the lysis buffer showed apparent bands of oxidized and reduced PTEN (Fig. 2A). In contrast, the bands of oxidized and reduced PTEN were not clear and blurred in the samples supplemented with lower than 1 mM NEM. Samples with 1 mM IAM and 10 mM IAM showed apparent bands of oxidized and reduced PTEN on the non-reducing SDS–PAGE gel. However, samples with 10 mM IAM showed no bands of PTEN on the reducing SDS–PAGE gel because 10 mM IAM caused the aggregation and precipitation of proteins immediately after mixing samples with reducing SDS–PAGE sample buffer (Fig. 2B). The bands of PTEN were not apparent in the samples supplemented with IAA (Fig. 2C).
Fig. 1. The scheme of experimental procedure for the assay of the redox state of PTEN by mobility shift.
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Fig. 2. Effect of NEM, IAM and IAA on the mobility of oxidized and reduced PTEN in HeLa cells. Cells were incubated in the absence or presence of 1 mM H2O2 for 5 min and then lysed with lysis buffer supplemented with various concentrations of NEM (A), IAM (B) and IAA (C) as indicated. Cell lysates (20 lg total protein) were resolved by nonreducing and reducing SDS–PAGE and subjected to Western blotting analysis with anti-PTEN and b-actin antibodies. b-actin levels were used as a loading control.
Next, we examined that oxidation of PTEN by H2O2 in cells at various concentrations and incubated for different times, after which cells were lysed with or without 10 mM NEM-supplemented lysis buffer and then subjected to immunoblot analysis with PTEN antibodies. Oxidized and reduced PTEN were apparently detected in the samples supplemented with 10 mM. The oxidized form of PTEN was increased in a dose-dependent manner and then declined. In contrast, in the samples without NEM, we did not identify oxidized PTEN bands (Fig. 3). These data suggested that supplementation of NEM in the lysis buffer and separation of lysate samples by non-reducing SDS–PAGE is crucial and effective to assess the redox state of PTEN. Previously, we showed that formation of oxidized PTEN band was abolished when Cys124 or Cys71 residues were replaced by Ser residue using recombinant wild type and mutant PTEN protein in vitro [10]. To examine the effects of mutation of Cys71 and Cys124 residues on the mobility shift of PTEN in cells, wild-type and mutant PTEN were expressed in NIH3T3 cells, which were treated with 0.7 mM H2O2 and incubated for the indicated times. Then, cell lysates were alkylated with 10 mM NEM and subjected to non-reducing SDS–PAGE and Western blotting with HA-specific antibodies. As a result, the oxidized PTEN band was not detected in the samples expressing Cys71 and Cys124 single mutant and Cys71/ Cys124 double mutant (Fig. 4). These results suggest that the
Cys71 and Cys124 are involved in the formation of an intramolecular disulfide bond that result in rapid migration of oxidized PTEN.
4. Concluding remarks PTEN is a well-known tumor suppressor. It is oxidized by ROS with the formation of an intracellular disulfide between the active site Cys124 and nearby Cys71 residues. PTEN oxidation inactivates the catalytic activity, causing elevation of AKT activity and downstream cascade signaling, resulting in deregulation of cell growth and proliferation. Oxidized PTEN is reversibly reduced over time after oxidative stress by H2O2. Cellular redox homeostasis is controlled by thioredoxin (Trx) and/or glutaredoxin (Grx) [17,19,20] Thus, changes in intracellular levels of these molecules can influence the redox status of PTEN. Therefore, redox regulation of PTEN play a key roles in tumorigenesis. Alkylating reagents are used to halt in the thiol–disulfide status in cells. N-ethylmaleimide (NEM), iodoacetamide (IAM), and iodoacetic acid (IAA) are commonly used for protein thiol alkylation. However, there is distinct difference in experimental approaches. IAA and IAM react irreversibly with thiols in nucleophilic substitution reactions to form the carboxymethyl or carboxamidomethyl derivatives. It is light sensitive and must be protected from lights during reactions. Moreover, to complete alkylation of protein thi-
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Fig. 3. Assay of redox state of PTEN with lysis buffer supplemented with NEM. Cells were treated with 1 mM H2O2 for the times indicated (0, 5, 10, 30, 60, 120 min) (A) and with various concentrations (0, 0.1, 0.5, 1, 2, 4 mM) for 10 min (B). Cells were lysed with lysis buffer supplemented with 10 mM NEM (right) or without NEM (left) and subjected to non-reducing and reducing SDS–PAGE followed by Western blot analysis.
Fig. 4. Effects of mutation of Cys71 or Cys124 residues on the mobility of PTEN. NIH3T3 cells expressing HA-tagged wild-type PTEN or C121S, C74S, C121/74S mutants were treated with 0.7 mM H2O2 for the times indicated (0, 2, 5, 10, 30, 90 min). Cells were lysed with lysis buffer supplemented with 10 mM NEM. Then, samples were subjected to non-reducing SDS–PAGE followed by Western blot analysis with a mouse anti-HA antibody.
ols, it is required for higher concentrations, pHs and reaction times. In contrast, the reaction of NEM with thiols is Michael-type addition reaction which across the double bond to form a thioether bond between the thiol and the maleimide. When compared with other alkylation reagent, the reaction of thiol alkylation with NEM requires less time and less dependent on pHs. Thus, NEM is more effective alkylation reagents than IAA, IAM and uncharged NEM can react in hydrophobic conditions [18,21]. In the previous studies for monitoring of intracellular thiols status of protein, cysteine residues are conjugated with alkylating reagents such as NEM, IAA and IAM that were biotin-tagged, fluorescently active, or radioactive [22–24]. Biotin labeling methods were subsequently measured by immunoblotting with streptavidin conjugated-HRP antibody. However, these methods are complicated and depend on reagent properties and on yield of reactions. In contrast, the gel mobility shift assay described in this report required only suitable antibody and NEM. In conclusion, the mobility shift assay using NEM as an alkylating agent, non-reduc-
ing SDS–PAGE and Western blotting with specific antibody was an effective and convenient method to assess redox state of PTEN in cells. This method might contribute to accelerate studies for identifying proteins involved in the redox regulation of PTEN in cells and disease models.
Acknowledgments This study was supported by a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No. HN11C0047).
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