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Muscarinic acetylcholine receptors regulate the dephosphorylation of eukaryotic translation elongation factor 2 in SNU-407 colon cancer cells
Biochemical and Biophysical Research Communications 516 (2019) 424e429
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Muscarinic acetylcholine receptors regulate the dephosphorylation of eukaryotic translation elongation factor 2 in SNU-407 colon cancer cells Bala Murali Krishna Vasamsetti 1, Ziyu Liu 1, Yang-Seo Park, Nam Jeong Cho* Department of Biochemistry, College of Natural Sciences, Biotechnology Research Institute, Chungbuk National University, Cheongju, 28644, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 May 2019 Accepted 11 June 2019 Available online 19 June 2019
Previously, we showed that muscarinic acetylcholine receptors (mAChRs) promote global protein biosynthesis in SNU-407 colon cancer cells. However, the molecular mechanisms underlying this event are poorly understood. Here, we asked whether mAChRs modulate the activity of eukaryotic translation elongation factor 2 (eEF2), which controls ribosomal translocation during the peptide elongation step. When SNU-407 cells were treated with the cholinergic agonist carbachol, eEF2 phosphorylation at T56 was decreased in a dose- and time-dependent manner. The muscarinic antagonist atropine almost completely blocked this effect of carbachol, demonstrating that mAChRs specifically regulate eEF2 dephosphorylation. We also investigated the signaling pathways that connect mAChR stimulation to eEF2 dephosphorylation using chemical inhibitors. Treating cells with U0126, a potent MEK1/2 inhibitor, decreased carbachol-stimulated eEF2 dephosphorylation. In contrast, the mTORC1 inhibitor rapamycin did not have a significant effect on eEF2 dephosphorylation. We also found that the protein kinase C (PKC) inhibitor GF109203X substantially reduced eEF2 dephosphorylation. Together, our experimental data indicate that the MEK1/2-ERK1/2 pathway and the PKC pathway, but not the mTORC1-S6K1 pathway, are involved in mAChR-mediated eEF2 dephosphorylation. © 2019 Elsevier Inc. All rights reserved.
Keywords: Muscarinic acetylcholine receptor eEF2 ERK1/2 PKC SNU-407 colon cancer cells Protein synthesis
1. Introduction Protein synthesis, also called translation, is an essential cellular process that controls cell growth and proliferation. A variety of factors are needed for translation, which occurs in three steps: initiation, elongation, and termination. One of the translation factors, eukaryotic translation elongation factor 2 (eEF2), is responsible for ribosomal translocation during the peptide elongation step. An increase in eEF2 phosphorylation inhibits its activity and reduces the rate of translation [1,2]. Overexpression of eEF2 has been observed in many different types of cancers, and knockdown of eEF2 inhibits cancer cell growth [3,4]. Conceivably, improper regulation of eEF2 activity results in abnormal cell growth and proliferation.
Muscarinic acetylcholine receptors (mAChRs), which are Gprotein coupled receptors, are known to mediate various nervous functions, including learning and memory [5,6]. In addition, mAChRs are implicated in cell growth and proliferation in diverse cell types [7e11]. Previously, we showed that mAChRs stimulate global protein biosynthesis in SNU-407 colon cancer cells and promote cell proliferation [12e14]. We observed that mAChRs induce eIF4B phosphorylation via the MEK1/2-ERK1/2 and protein kinase C (PKC) signaling pathways [15] and presumably activate translation initiation. However, it is not known whether translation elongation is also controlled by mAChRs in SNU-407 cells. In the current work, we provide evidence that mAChRs decrease eEF2 phosphorylation via the MEK1/2-ERK1/2 and PKC pathways.
Abbreviations: eEF2, eukaryotic translation elongation factor 2; ERK1/2, extracellular signal-regulated kinases 1/2; mAChR, muscarinic acetylcholine receptor; mTORC1, mammalian target of rapamycin complex 1; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; PP2A, protein phosphatase 2A; S6K1, ribosomal protein S6 kinase 1. * Corresponding author. Department of Biochemistry, College of Natural Sciences, Chungbuk National University, Cheongju, 28644, South Korea. E-mail address: [email protected] (N.J. Cho). 1 These two authors contributed equally to this work. https://doi.org/10.1016/j.bbrc.2019.06.059 0006-291X/© 2019 Elsevier Inc. All rights reserved.
B.M.K. Vasamsetti et al. / Biochemical and Biophysical Research Communications 516 (2019) 424e429
2. Materials and methods 2.1. Materials GF109203X, rapamycin and okadaic acid were obtained from Tocris Bioscience. EDTA and EGTA were from Amresco. All other chemicals were purchased from Sigma-Aldrich. Antibodies used in the study: The following antibodies were used in the experiments: phospho-eEF2 (T56) antibody (#2331, Cell Signaling Technology); eEF2 antibody (#2332, Cell Signaling Technology); phospho-S6K1 (T389) antibody (#9234, Cell Signaling Technology); phospho-ERK1/2 antibody (#4370, Cell Signaling Technology); and anti-tubulin antibody (#2125, Cell Signaling Technology). 2.2. Cell culture The SNU-407 colon cancer cell line was obtained from the Korean Cell Line Bank (Seoul, Korea). The cells were grown in RPMI
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medium 1640 (Gibco) supplemented with 10% FBS (Gibco) and antibiotics (Gibco) (100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B) and were cultured at 37 C in 5e6% CO2. 2.3. Western blot analysis SNU-407 colon cancer cells were grown in 12-well plates for 20e24 h, serum-starved for 18e24 h, and treated with carbachol in serum-free RPMI 1640 at 37 C. The cells were washed twice with ice-cold phosphate-buffered saline (PBS) and incubated with lysis buffer (20 mM HEPES at pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0.5% Nonidet P-40, and 10% glycerol) for 1 h at 4 C. The cells were collected and centrifuged at 15,000g for 30 min at 4 C. The supernatant was boiled for 5 min in SDS sample buffer, separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were incubated for 1 h with blocking buffer (20 mM Tris-HCl at pH 7.6, 140 mM NaCl, 0.1% Tween-20, and 5% skim milk)
Fig. 1. mAChRs mediate eEF2 dephosphorylation. (A) SNU-407 cells were serum-starved for 18e24 h and treated with various concentrations of carbachol for 30 min. (B) Cells were serum-starved for 18e24 h and treated with 0.1 mM carbachol for the indicated times. (C) Cells were serum-starved for 18e24 h and pretreated with 1 mM atropine for 5 min before stimulation with 0.1 mM carbachol for 5 or 30 min. After treatment, the cell lysates were immunoblotted with antibodies to detect phospho-eEF2 (T56) or total eEF2. A representative western blot is shown in the upper panel, and the eEF2 phosphorylation data (mean ± SEM, n ¼ 3) are shown in the lower panel. **P < 0.01 by paired t-test.
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at room temperature and treated with primary antibodies overnight at 4 C. The membranes were then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (#7074, Cell Signaling Technology) for 1e1.5 h at room temperature, and the immunoreactive bands were visualized using Clarity Western ECL Substrate (Bio-Rad). The densities of the bands were analyzed using ImageJ (NIH). 2.4. Statistical analysis The data were analyzed using GraphPad Prism 6 (GraphPad Software). Statistical significance was determined using a paired ttest or two-way repeated measures (RM) analysis of variance (ANOVA). P-values less than 0.05 were considered significant. 3. Results 3.1. mAChRs mediate eEF2 dephosphorylation To understand the molecular mechanism by which mAChRs promote global protein biosynthesis, we examined whether mAChRs modulate the phosphorylation status of eEF2, a key regulator of protein biosynthesis. For this purpose, we performed western blot experiments with an antibody specific to phosphoeEF2 (T56). Previous studies have established that eEF2 dephosphorylation at T56 is closely associated with increased translational activity [16,17]. When SNU-407 colon cancer cells were treated with the cholinergic agonist carbachol, eEF2 phosphorylation was dose-dependently decreased (Fig. 1A). eEF2 dephosphorylation was evident at 1 mM carbachol, and a minimal level of eEF2 phosphorylation was observed at 0.1 mM carbachol. A time-dependent decrease in eEF2 phosphorylation was also observed in the presence of 0.1 M carbachol (Fig. 1B). eEF2 phosphorylation decreased rapidly (within 5 min) and reached its minimum at 15 min. To verify that carbachol-stimulated eEF2 dephosphorylation was mediated by mAChRs, we pretreated the cells with the muscarinic antagonist atropine before carbachol stimulation. Atropine treatment almost completely blocked the carbachol effect (Fig. 1C). Overall, our data demonstrate that mAChRs induce eEF2 dephosphorylation at T56 in SNU-407 colon cancer cells. 3.2. mAChR-mediated eEF2 dephosphorylation depends on the MEK1/2-ERK1/2 pathway, but not on the mTORC1-S6K1 pathway Previously, we showed that mAChRs promote global protein biosynthesis via both the MEK1/2-ERK1/2 and mTORC1-S6K1 pathways in SNU-407 cells [14]. To determine whether these signaling pathways are involved in mAChR-mediated eEF2 dephosphorylation, we used the MEK1/2 inhibitor U0126 and the mTORC1 inhibitor rapamycin. Treating cells with U0126 substantially interfered with carbachol-stimulated eEF2 dephosphorylation (Fig. 2), indicating that the MEK1/2-ERK1/2 pathway is required for linking mAChR stimulation to eEF2 dephosphorylation. In contrast, rapamycin treatment did not significantly diminish carbacholstimulated eEF2 dephosphorylation (Fig. 2). The same dose of rapamycin abrogated carbachol-induced S6K1 phosphorylation. These results imply that the mTORC1-S6K1 pathway does not have a role in mAChR-mediated eEF2 dephosphorylation in SNU-407 cells. 3.3. PKC is involved in mAChR-mediated eEF2 dephosphorylation We previously observed that PKC is important for carbacholstimulated ERK1/2 activation and proliferation in SNU-407 colon cancer cells [13]. We thus asked whether PKC is involved in mAChRmediated eEF2 dephosphorylation. When SNU-407 cells were
Fig. 2. The MEK1/2-ERK1/2 pathway, but not the mTORC1-S6K1 pathway, is involved in mAChR-mediated eEF2 dephosphorylation. SNU-407 cells were serum-starved for 18e24 h and pretreated with 1 mM U0126 (MEK1/2 inhibitor) or 20 nM rapamycin (mTORC1 inhibitor) for 30 min before stimulation with 0.1 mM carbachol for 5 or 30 min. The cell lysates were immunoblotted with antibodies to detect phospho-eEF2 (T56), phospho-S6K1, phospho-ERK1/2, or tubulin. A representative western blot is shown in the upper panel, and the eEF2 phosphorylation data (mean ± SEM, n ¼ 4) are shown in the lower panel. The carbachol-induced difference in eEF2 phosphorylation was analyzed by two-way RM ANOVA. *P < 0.05.
pretreated with the PKC inhibitor GF109203X, carbacholstimulated eEF2 dephosphorylation was significantly diminished (Fig. 3A). These results suggest that PKC participates in the mAChRmediated decrease in eEF2 phosphorylation. To test whether PKC stimulation alone elicits eEF2 dephosphorylation, we treated SNU-407 colon cancer cells with the PKC activator phorbol 12-myristate 13-acetate (PMA). PMA treatment for a short period of time (15 min) substantially induced eEF2 dephosphorylation (Fig. 3B), indicating that PKC activation can induce eEF2 dephosphorylation. Notably, the effect of PMA treatment on eEF2 dephosphorylation was weaker than that of carbachol treatment, although ERK1/2 phosphorylation induced by PMA treatment was much greater than that induced by carbachol treatment. These results raise the possibility that another signaling pathway, in addition to the MEK1/2-ERK1/2 and PKC pathways, is involved in carbachol-stimulated eEF2 dephosphorylation. To verify the function of PKC in mAChR-mediated eEF2 dephosphorylation, we pretreated the cells with PMA for a long time (overnight) to deplete PKC activity. Carbachol-stimulated eEF2 dephosphorylation was almost completely blocked in the PKCdepleted cells (Fig. 3C), further supporting the notion that PKC is necessary for mAChR-mediated eEF2 dephosphorylation in SNU407 colon cancer cells. 3.4. Protein phosphatases are involved in mAChR-mediated eEF2 dephosphorylation Diverse types of protein phosphatases are present in eukaryotic cells to dephosphorylate phosphorylated proteins. To obtain information about the protein phosphatases regulated by mAChRs for eEF2 dephosphorylation, we first tested the effect of sodium fluoride (NaF), a general inhibitor of Ser/Thr and acidic phosphatases. As shown in Fig. 4A, NaF treatment abolished carbachol-stimulated
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Fig. 3. mAChR-mediated eEF2 dephosphorylation is PKC-dependent. (A) SNU-407 cells were serum-starved for 18e24 h and pretreated with 1 mM GF09203X (PKC inhibitor) before stimulation with 0.1 mM carbachol for 5 or 30 min. The cell lysates were immunoblotted with antibodies to detect phospho-eEF2 (T56) or tubulin. A representative western blot is shown in the upper panel, and the eEF2 phosphorylation data (mean ± SEM, n ¼ 4) are shown in the lower panel. The carbachol-induced difference in eEF2 phosphorylation was analyzed by two-way RM ANOVA. *P < 0.05. (B) Cells were serum-starved for 18e24 h and treated with 0.1 mM carbachol or 1 mM PMA for 15 min. The cell lysates were immunoblotted with antibodies to detect phospho-eEF2 (T56), phospho-ERK1/2, or tubulin. A representative western blot is shown in the upper panel, and the eEF2 phosphorylation data (mean ± SEM, n ¼ 3) are shown in the lower panel. ***P < 0.001 and *P < 0.05 by paired t-test. (C) Cells were serum-starved for 18e24 h and incubated with 1 mM PMA overnight (o/n) before stimulation with 0.1 mM carbachol for 5 or 30 min. The cell lysates were immunoblotted with antibodies to detect phospho-eEF2 (T56) or tubulin. A representative western blot is shown in the upper panel, and the eEF2 phosphorylation data (mean ± SEM, n ¼ 4) are shown in the lower panel. The carbachol-induced difference in eEF2 phosphorylation was analyzed by two-way RM ANOVA. *P < 0.05.
eEF2 dephosphorylation, demonstrating the involvement of Ser/ Thr and/or acidic phosphatase(s). We observed that the basal level of eEF2 phosphorylation, in the absence of carbachol, was markedly increased by NaF treatment, which indicates that an unidentified, non-cholinergic system plays a role in eEF2 dephosphorylation at T56 in SNU-407 colon cancer cells. We next asked whether protein phosphatase 2A (PP2A) is important for mAChR-mediated eEF2 dephosphorylation in SNU407 cells. PP2A has been reported to participate in eEF2 dephosphorylation in various cell types [18e20]. For this purpose, we treated the cells with okadaic acid, a potent PP2A inhibitor [21,22],
before stimulation with carbachol. As illustrated in Fig. 4B, okadaic acid treatment abrogated the carbachol effect at 5 min but only slightly altered the carbachol effect at 30 min. These results imply that while the early phase of carbachol-stimulated eEF2 dephosphorylation is mediated by PP2A, the late phase of eEF2 dephosphorylation is mediated largely by another protein phosphatase, which is yet to be determined. 4. Discussion Recently, we found that mAChRs promote global protein
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Fig. 4. Protein phosphatases are involved in mAChR-mediated eEF2 dephosphorylation. (A) SNU-407 cells were serum-starved for 18e24 h and pretreated with 20 mM sodium fluoride (NaF) for 30 min before stimulation with 0.1 mM carbachol for 5 or 30 min. (B) Cells were serum-starved for 18e24 h and pretreated with 0.5 mM okadaic acid (PP2A inhibitor) for 30 min before stimulation with 0.1 mM carbachol for 5 or 30 min. The cell lysates were immunoblotted with antibodies to detect phospho-eEF2 (T56) or tubulin. A representative western blot is shown in the upper panel, and the eEF2 phosphorylation data (mean ± SEM, n ¼ 4) are shown in the lower panel. The carbachol-induced difference in eEF2 phosphorylation was analyzed by two-way RM ANOVA. **P < 0.01; *P < 0.05.
biosynthesis in SNU-407 colon cancer cells [14]. In an attempt to understand the molecular mechanisms underlying this event, we investigated whether mAChRs regulate the phosphorylation status of eEF2, a key element of the translation elongation machinery. Experimental results indicate that mAChRs mediate eEF2 dephosphorylation and presumably activate translation elongation. We previously reported that mAChRs induce the phosphorylation of eIF4B, an important factor for translation initiation [15]. Therefore, mAChRs appear to modulate protein synthesis at the level of both translation initiation and elongation. Our previous data revealed that mAChRs enhance global protein biosynthesis and cell proliferation via both the MEK1/2-ERK1/2 and mTORC1-S6K1 pathways [14]. Our current data indicate that the MEK1/2-ERK1/2 pathway, but not the mTORC1-S6K1 pathway, is involved in mAChR-mediated eEF2 dephosphorylation. In contrast, mAChR-mediated phosphorylation of 4E-BP1, an essential component of translation initiation, occurs via the mTORC1-S6K1 pathway but not through the MEK1/2-ERK1/2 pathway (manuscript in preparation). Overall, the two signaling pathways induced by mAChRs have distinct roles in controlling translation. In response to various stimuli, diverse signaling pathways are utilized for eEF2 dephosphorylation. For instance, the decrease in eEF2 phosphorylation induced by insulin, cholecystokinin or growth hormone is reduced by rapamycin treatment [23e25]. In comparison, oxytocin-stimulated eEF2 dephosphorylation is resistant to U0126 and rapamycin but sensitive to the PKC inhibitor Go6983 [26]. Thus, it can be postulated that the signaling pathways underlying eEF2 dephosphorylation depend on the type of extracellular signals. We cannot exclude the possibility, however, that the signaling pathways might depend on the cell type. Upon stimulation, the temporal activation profiles of separate
signaling pathways are considered to be different. For example, in SNU-407 cells, ERK1/2 activity peaks at 5 min of carbachol stimulation and begins to decrease after 30 min [13], whereas S6K1 activity increase slowly after 5 min of carbachol stimulation and peaks at 30 min [14]. When we tested the effects of chemical inhibitors on mAChR-mediated eEF2 dephosphorylation, we chose two time points, 5 min and 30 min, to obtain better information about the signaling pathways underlying this process. The inhibitory effect of U0126 on carbachol-stimulated eEF2 dephosphorylation was stronger at 5 min than at 30 min (Fig. 2), consistent with the idea that the MEK1/2-ERK1/2 pathway plays an important role in eEF2 dephosphorylation. Similarly, the effect of okadaic acid on eEF2 dephosphorylation was much stronger at 5 min than at 30 min (Fig. 4B), suggesting that while PP2A is mainly responsible for the early phase of eEF2 dephosphorylation, okadaic acid-resistant protein phosphatase(s) may be involved in the late phase. The effect of GF109203X on mAChR-mediated eEF2 dephosphorylation was robust at 5 min of carbachol stimulation but rather weak at 30 min of carbachol stimulation (Fig. 3A). In contrast, longterm PMA treatment resulted in near-complete inhibition of eEF2 dephosphorylation at both 5 and 30 min of carbachol stimulation (Fig. 3C). We speculate that the PKC isoforms affected by GF109203X and those depleted by long-term PMA treatment may be overlapping but not identical in SNU-407 cells. Conceivably, the GF109203X-sensitive and GF109203Xeresistant PKC isoforms serve their respective functions in the early and late phases of carbachol-stimulated eEF2 dephosphorylation. In summary, we provide evidence that mAChRs mediate eEF2 dephosphorylation in SNU-407 colon cancer cells via the MEK1/2ERK1/2 pathway but not the mTORC1-S6K1 pathway. We also showed that PKC is involved in mAChR-mediated eEF2
B.M.K. Vasamsetti et al. / Biochemical and Biophysical Research Communications 516 (2019) 424e429
dephosphorylation. It appears that PP2A participates in linking the stimulation of mAChRs to the activation of eEF2.
[10]
Conflicts of interest [11]
The authors declare no conflict of interest. [12]
Acknowledgements
[13]
This work was supported by the Basic Research Program through the National Research Foundation of Korea (2013R1A1A2058625 and 2017R1D1A3B03030588).
[14]
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Muscarinic acetylcholine receptors regulate the dephosphorylation of eukaryotic translation elongation factor 2 in SNU-407 colon cancer cells Bala Murali Krishna Vasamsetti 1, Ziyu Liu 1, Yang-Seo Park, Nam Jeong Cho* Department of Biochemistry, College of Natural Sciences, Biotechnology Research Institute, Chungbuk National University, Cheongju, 28644, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 May 2019 Accepted 11 June 2019 Available online 19 June 2019
Previously, we showed that muscarinic acetylcholine receptors (mAChRs) promote global protein biosynthesis in SNU-407 colon cancer cells. However, the molecular mechanisms underlying this event are poorly understood. Here, we asked whether mAChRs modulate the activity of eukaryotic translation elongation factor 2 (eEF2), which controls ribosomal translocation during the peptide elongation step. When SNU-407 cells were treated with the cholinergic agonist carbachol, eEF2 phosphorylation at T56 was decreased in a dose- and time-dependent manner. The muscarinic antagonist atropine almost completely blocked this effect of carbachol, demonstrating that mAChRs specifically regulate eEF2 dephosphorylation. We also investigated the signaling pathways that connect mAChR stimulation to eEF2 dephosphorylation using chemical inhibitors. Treating cells with U0126, a potent MEK1/2 inhibitor, decreased carbachol-stimulated eEF2 dephosphorylation. In contrast, the mTORC1 inhibitor rapamycin did not have a significant effect on eEF2 dephosphorylation. We also found that the protein kinase C (PKC) inhibitor GF109203X substantially reduced eEF2 dephosphorylation. Together, our experimental data indicate that the MEK1/2-ERK1/2 pathway and the PKC pathway, but not the mTORC1-S6K1 pathway, are involved in mAChR-mediated eEF2 dephosphorylation. © 2019 Elsevier Inc. All rights reserved.
Keywords: Muscarinic acetylcholine receptor eEF2 ERK1/2 PKC SNU-407 colon cancer cells Protein synthesis
1. Introduction Protein synthesis, also called translation, is an essential cellular process that controls cell growth and proliferation. A variety of factors are needed for translation, which occurs in three steps: initiation, elongation, and termination. One of the translation factors, eukaryotic translation elongation factor 2 (eEF2), is responsible for ribosomal translocation during the peptide elongation step. An increase in eEF2 phosphorylation inhibits its activity and reduces the rate of translation [1,2]. Overexpression of eEF2 has been observed in many different types of cancers, and knockdown of eEF2 inhibits cancer cell growth [3,4]. Conceivably, improper regulation of eEF2 activity results in abnormal cell growth and proliferation.
Muscarinic acetylcholine receptors (mAChRs), which are Gprotein coupled receptors, are known to mediate various nervous functions, including learning and memory [5,6]. In addition, mAChRs are implicated in cell growth and proliferation in diverse cell types [7e11]. Previously, we showed that mAChRs stimulate global protein biosynthesis in SNU-407 colon cancer cells and promote cell proliferation [12e14]. We observed that mAChRs induce eIF4B phosphorylation via the MEK1/2-ERK1/2 and protein kinase C (PKC) signaling pathways [15] and presumably activate translation initiation. However, it is not known whether translation elongation is also controlled by mAChRs in SNU-407 cells. In the current work, we provide evidence that mAChRs decrease eEF2 phosphorylation via the MEK1/2-ERK1/2 and PKC pathways.
Abbreviations: eEF2, eukaryotic translation elongation factor 2; ERK1/2, extracellular signal-regulated kinases 1/2; mAChR, muscarinic acetylcholine receptor; mTORC1, mammalian target of rapamycin complex 1; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; PP2A, protein phosphatase 2A; S6K1, ribosomal protein S6 kinase 1. * Corresponding author. Department of Biochemistry, College of Natural Sciences, Chungbuk National University, Cheongju, 28644, South Korea. E-mail address: [email protected] (N.J. Cho). 1 These two authors contributed equally to this work. https://doi.org/10.1016/j.bbrc.2019.06.059 0006-291X/© 2019 Elsevier Inc. All rights reserved.
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2. Materials and methods 2.1. Materials GF109203X, rapamycin and okadaic acid were obtained from Tocris Bioscience. EDTA and EGTA were from Amresco. All other chemicals were purchased from Sigma-Aldrich. Antibodies used in the study: The following antibodies were used in the experiments: phospho-eEF2 (T56) antibody (#2331, Cell Signaling Technology); eEF2 antibody (#2332, Cell Signaling Technology); phospho-S6K1 (T389) antibody (#9234, Cell Signaling Technology); phospho-ERK1/2 antibody (#4370, Cell Signaling Technology); and anti-tubulin antibody (#2125, Cell Signaling Technology). 2.2. Cell culture The SNU-407 colon cancer cell line was obtained from the Korean Cell Line Bank (Seoul, Korea). The cells were grown in RPMI
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medium 1640 (Gibco) supplemented with 10% FBS (Gibco) and antibiotics (Gibco) (100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B) and were cultured at 37 C in 5e6% CO2. 2.3. Western blot analysis SNU-407 colon cancer cells were grown in 12-well plates for 20e24 h, serum-starved for 18e24 h, and treated with carbachol in serum-free RPMI 1640 at 37 C. The cells were washed twice with ice-cold phosphate-buffered saline (PBS) and incubated with lysis buffer (20 mM HEPES at pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0.5% Nonidet P-40, and 10% glycerol) for 1 h at 4 C. The cells were collected and centrifuged at 15,000g for 30 min at 4 C. The supernatant was boiled for 5 min in SDS sample buffer, separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were incubated for 1 h with blocking buffer (20 mM Tris-HCl at pH 7.6, 140 mM NaCl, 0.1% Tween-20, and 5% skim milk)
Fig. 1. mAChRs mediate eEF2 dephosphorylation. (A) SNU-407 cells were serum-starved for 18e24 h and treated with various concentrations of carbachol for 30 min. (B) Cells were serum-starved for 18e24 h and treated with 0.1 mM carbachol for the indicated times. (C) Cells were serum-starved for 18e24 h and pretreated with 1 mM atropine for 5 min before stimulation with 0.1 mM carbachol for 5 or 30 min. After treatment, the cell lysates were immunoblotted with antibodies to detect phospho-eEF2 (T56) or total eEF2. A representative western blot is shown in the upper panel, and the eEF2 phosphorylation data (mean ± SEM, n ¼ 3) are shown in the lower panel. **P < 0.01 by paired t-test.
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at room temperature and treated with primary antibodies overnight at 4 C. The membranes were then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (#7074, Cell Signaling Technology) for 1e1.5 h at room temperature, and the immunoreactive bands were visualized using Clarity Western ECL Substrate (Bio-Rad). The densities of the bands were analyzed using ImageJ (NIH). 2.4. Statistical analysis The data were analyzed using GraphPad Prism 6 (GraphPad Software). Statistical significance was determined using a paired ttest or two-way repeated measures (RM) analysis of variance (ANOVA). P-values less than 0.05 were considered significant. 3. Results 3.1. mAChRs mediate eEF2 dephosphorylation To understand the molecular mechanism by which mAChRs promote global protein biosynthesis, we examined whether mAChRs modulate the phosphorylation status of eEF2, a key regulator of protein biosynthesis. For this purpose, we performed western blot experiments with an antibody specific to phosphoeEF2 (T56). Previous studies have established that eEF2 dephosphorylation at T56 is closely associated with increased translational activity [16,17]. When SNU-407 colon cancer cells were treated with the cholinergic agonist carbachol, eEF2 phosphorylation was dose-dependently decreased (Fig. 1A). eEF2 dephosphorylation was evident at 1 mM carbachol, and a minimal level of eEF2 phosphorylation was observed at 0.1 mM carbachol. A time-dependent decrease in eEF2 phosphorylation was also observed in the presence of 0.1 M carbachol (Fig. 1B). eEF2 phosphorylation decreased rapidly (within 5 min) and reached its minimum at 15 min. To verify that carbachol-stimulated eEF2 dephosphorylation was mediated by mAChRs, we pretreated the cells with the muscarinic antagonist atropine before carbachol stimulation. Atropine treatment almost completely blocked the carbachol effect (Fig. 1C). Overall, our data demonstrate that mAChRs induce eEF2 dephosphorylation at T56 in SNU-407 colon cancer cells. 3.2. mAChR-mediated eEF2 dephosphorylation depends on the MEK1/2-ERK1/2 pathway, but not on the mTORC1-S6K1 pathway Previously, we showed that mAChRs promote global protein biosynthesis via both the MEK1/2-ERK1/2 and mTORC1-S6K1 pathways in SNU-407 cells [14]. To determine whether these signaling pathways are involved in mAChR-mediated eEF2 dephosphorylation, we used the MEK1/2 inhibitor U0126 and the mTORC1 inhibitor rapamycin. Treating cells with U0126 substantially interfered with carbachol-stimulated eEF2 dephosphorylation (Fig. 2), indicating that the MEK1/2-ERK1/2 pathway is required for linking mAChR stimulation to eEF2 dephosphorylation. In contrast, rapamycin treatment did not significantly diminish carbacholstimulated eEF2 dephosphorylation (Fig. 2). The same dose of rapamycin abrogated carbachol-induced S6K1 phosphorylation. These results imply that the mTORC1-S6K1 pathway does not have a role in mAChR-mediated eEF2 dephosphorylation in SNU-407 cells. 3.3. PKC is involved in mAChR-mediated eEF2 dephosphorylation We previously observed that PKC is important for carbacholstimulated ERK1/2 activation and proliferation in SNU-407 colon cancer cells [13]. We thus asked whether PKC is involved in mAChRmediated eEF2 dephosphorylation. When SNU-407 cells were
Fig. 2. The MEK1/2-ERK1/2 pathway, but not the mTORC1-S6K1 pathway, is involved in mAChR-mediated eEF2 dephosphorylation. SNU-407 cells were serum-starved for 18e24 h and pretreated with 1 mM U0126 (MEK1/2 inhibitor) or 20 nM rapamycin (mTORC1 inhibitor) for 30 min before stimulation with 0.1 mM carbachol for 5 or 30 min. The cell lysates were immunoblotted with antibodies to detect phospho-eEF2 (T56), phospho-S6K1, phospho-ERK1/2, or tubulin. A representative western blot is shown in the upper panel, and the eEF2 phosphorylation data (mean ± SEM, n ¼ 4) are shown in the lower panel. The carbachol-induced difference in eEF2 phosphorylation was analyzed by two-way RM ANOVA. *P < 0.05.
pretreated with the PKC inhibitor GF109203X, carbacholstimulated eEF2 dephosphorylation was significantly diminished (Fig. 3A). These results suggest that PKC participates in the mAChRmediated decrease in eEF2 phosphorylation. To test whether PKC stimulation alone elicits eEF2 dephosphorylation, we treated SNU-407 colon cancer cells with the PKC activator phorbol 12-myristate 13-acetate (PMA). PMA treatment for a short period of time (15 min) substantially induced eEF2 dephosphorylation (Fig. 3B), indicating that PKC activation can induce eEF2 dephosphorylation. Notably, the effect of PMA treatment on eEF2 dephosphorylation was weaker than that of carbachol treatment, although ERK1/2 phosphorylation induced by PMA treatment was much greater than that induced by carbachol treatment. These results raise the possibility that another signaling pathway, in addition to the MEK1/2-ERK1/2 and PKC pathways, is involved in carbachol-stimulated eEF2 dephosphorylation. To verify the function of PKC in mAChR-mediated eEF2 dephosphorylation, we pretreated the cells with PMA for a long time (overnight) to deplete PKC activity. Carbachol-stimulated eEF2 dephosphorylation was almost completely blocked in the PKCdepleted cells (Fig. 3C), further supporting the notion that PKC is necessary for mAChR-mediated eEF2 dephosphorylation in SNU407 colon cancer cells. 3.4. Protein phosphatases are involved in mAChR-mediated eEF2 dephosphorylation Diverse types of protein phosphatases are present in eukaryotic cells to dephosphorylate phosphorylated proteins. To obtain information about the protein phosphatases regulated by mAChRs for eEF2 dephosphorylation, we first tested the effect of sodium fluoride (NaF), a general inhibitor of Ser/Thr and acidic phosphatases. As shown in Fig. 4A, NaF treatment abolished carbachol-stimulated
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Fig. 3. mAChR-mediated eEF2 dephosphorylation is PKC-dependent. (A) SNU-407 cells were serum-starved for 18e24 h and pretreated with 1 mM GF09203X (PKC inhibitor) before stimulation with 0.1 mM carbachol for 5 or 30 min. The cell lysates were immunoblotted with antibodies to detect phospho-eEF2 (T56) or tubulin. A representative western blot is shown in the upper panel, and the eEF2 phosphorylation data (mean ± SEM, n ¼ 4) are shown in the lower panel. The carbachol-induced difference in eEF2 phosphorylation was analyzed by two-way RM ANOVA. *P < 0.05. (B) Cells were serum-starved for 18e24 h and treated with 0.1 mM carbachol or 1 mM PMA for 15 min. The cell lysates were immunoblotted with antibodies to detect phospho-eEF2 (T56), phospho-ERK1/2, or tubulin. A representative western blot is shown in the upper panel, and the eEF2 phosphorylation data (mean ± SEM, n ¼ 3) are shown in the lower panel. ***P < 0.001 and *P < 0.05 by paired t-test. (C) Cells were serum-starved for 18e24 h and incubated with 1 mM PMA overnight (o/n) before stimulation with 0.1 mM carbachol for 5 or 30 min. The cell lysates were immunoblotted with antibodies to detect phospho-eEF2 (T56) or tubulin. A representative western blot is shown in the upper panel, and the eEF2 phosphorylation data (mean ± SEM, n ¼ 4) are shown in the lower panel. The carbachol-induced difference in eEF2 phosphorylation was analyzed by two-way RM ANOVA. *P < 0.05.
eEF2 dephosphorylation, demonstrating the involvement of Ser/ Thr and/or acidic phosphatase(s). We observed that the basal level of eEF2 phosphorylation, in the absence of carbachol, was markedly increased by NaF treatment, which indicates that an unidentified, non-cholinergic system plays a role in eEF2 dephosphorylation at T56 in SNU-407 colon cancer cells. We next asked whether protein phosphatase 2A (PP2A) is important for mAChR-mediated eEF2 dephosphorylation in SNU407 cells. PP2A has been reported to participate in eEF2 dephosphorylation in various cell types [18e20]. For this purpose, we treated the cells with okadaic acid, a potent PP2A inhibitor [21,22],
before stimulation with carbachol. As illustrated in Fig. 4B, okadaic acid treatment abrogated the carbachol effect at 5 min but only slightly altered the carbachol effect at 30 min. These results imply that while the early phase of carbachol-stimulated eEF2 dephosphorylation is mediated by PP2A, the late phase of eEF2 dephosphorylation is mediated largely by another protein phosphatase, which is yet to be determined. 4. Discussion Recently, we found that mAChRs promote global protein
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Fig. 4. Protein phosphatases are involved in mAChR-mediated eEF2 dephosphorylation. (A) SNU-407 cells were serum-starved for 18e24 h and pretreated with 20 mM sodium fluoride (NaF) for 30 min before stimulation with 0.1 mM carbachol for 5 or 30 min. (B) Cells were serum-starved for 18e24 h and pretreated with 0.5 mM okadaic acid (PP2A inhibitor) for 30 min before stimulation with 0.1 mM carbachol for 5 or 30 min. The cell lysates were immunoblotted with antibodies to detect phospho-eEF2 (T56) or tubulin. A representative western blot is shown in the upper panel, and the eEF2 phosphorylation data (mean ± SEM, n ¼ 4) are shown in the lower panel. The carbachol-induced difference in eEF2 phosphorylation was analyzed by two-way RM ANOVA. **P < 0.01; *P < 0.05.
biosynthesis in SNU-407 colon cancer cells [14]. In an attempt to understand the molecular mechanisms underlying this event, we investigated whether mAChRs regulate the phosphorylation status of eEF2, a key element of the translation elongation machinery. Experimental results indicate that mAChRs mediate eEF2 dephosphorylation and presumably activate translation elongation. We previously reported that mAChRs induce the phosphorylation of eIF4B, an important factor for translation initiation [15]. Therefore, mAChRs appear to modulate protein synthesis at the level of both translation initiation and elongation. Our previous data revealed that mAChRs enhance global protein biosynthesis and cell proliferation via both the MEK1/2-ERK1/2 and mTORC1-S6K1 pathways [14]. Our current data indicate that the MEK1/2-ERK1/2 pathway, but not the mTORC1-S6K1 pathway, is involved in mAChR-mediated eEF2 dephosphorylation. In contrast, mAChR-mediated phosphorylation of 4E-BP1, an essential component of translation initiation, occurs via the mTORC1-S6K1 pathway but not through the MEK1/2-ERK1/2 pathway (manuscript in preparation). Overall, the two signaling pathways induced by mAChRs have distinct roles in controlling translation. In response to various stimuli, diverse signaling pathways are utilized for eEF2 dephosphorylation. For instance, the decrease in eEF2 phosphorylation induced by insulin, cholecystokinin or growth hormone is reduced by rapamycin treatment [23e25]. In comparison, oxytocin-stimulated eEF2 dephosphorylation is resistant to U0126 and rapamycin but sensitive to the PKC inhibitor Go6983 [26]. Thus, it can be postulated that the signaling pathways underlying eEF2 dephosphorylation depend on the type of extracellular signals. We cannot exclude the possibility, however, that the signaling pathways might depend on the cell type. Upon stimulation, the temporal activation profiles of separate
signaling pathways are considered to be different. For example, in SNU-407 cells, ERK1/2 activity peaks at 5 min of carbachol stimulation and begins to decrease after 30 min [13], whereas S6K1 activity increase slowly after 5 min of carbachol stimulation and peaks at 30 min [14]. When we tested the effects of chemical inhibitors on mAChR-mediated eEF2 dephosphorylation, we chose two time points, 5 min and 30 min, to obtain better information about the signaling pathways underlying this process. The inhibitory effect of U0126 on carbachol-stimulated eEF2 dephosphorylation was stronger at 5 min than at 30 min (Fig. 2), consistent with the idea that the MEK1/2-ERK1/2 pathway plays an important role in eEF2 dephosphorylation. Similarly, the effect of okadaic acid on eEF2 dephosphorylation was much stronger at 5 min than at 30 min (Fig. 4B), suggesting that while PP2A is mainly responsible for the early phase of eEF2 dephosphorylation, okadaic acid-resistant protein phosphatase(s) may be involved in the late phase. The effect of GF109203X on mAChR-mediated eEF2 dephosphorylation was robust at 5 min of carbachol stimulation but rather weak at 30 min of carbachol stimulation (Fig. 3A). In contrast, longterm PMA treatment resulted in near-complete inhibition of eEF2 dephosphorylation at both 5 and 30 min of carbachol stimulation (Fig. 3C). We speculate that the PKC isoforms affected by GF109203X and those depleted by long-term PMA treatment may be overlapping but not identical in SNU-407 cells. Conceivably, the GF109203X-sensitive and GF109203Xeresistant PKC isoforms serve their respective functions in the early and late phases of carbachol-stimulated eEF2 dephosphorylation. In summary, we provide evidence that mAChRs mediate eEF2 dephosphorylation in SNU-407 colon cancer cells via the MEK1/2ERK1/2 pathway but not the mTORC1-S6K1 pathway. We also showed that PKC is involved in mAChR-mediated eEF2
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dephosphorylation. It appears that PP2A participates in linking the stimulation of mAChRs to the activation of eEF2.
[10]
Conflicts of interest [11]
The authors declare no conflict of interest. [12]
Acknowledgements
[13]
This work was supported by the Basic Research Program through the National Research Foundation of Korea (2013R1A1A2058625 and 2017R1D1A3B03030588).
[14]
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