ERK1 phosphorylates Nanog to regulate protein stability and ... - Core

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Apr 13, 2014 - Zigang Dong a,⁎ a The Hormel Institute, University of Minnesota, 801, 16th AVE, NE, Austin, MN 55912, USA b Kyungpook National University, ...
Stem Cell Research (2014) 13, 1–11

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ERK1 phosphorylates Nanog to regulate protein stability and stem cell self-renewal Sung-Hyun Kim a,b,1 , Myoung Ok Kim a,b,1 , Yong-Yeon Cho a,2 , Ke Yao a , Dong Joon Kim a , Chul-Ho Jeong a , Dong Hoon Yu a , Ki Beom Bae a , Eun Jin Cho a , Sung Keun Jung a , Mee Hyun Lee a , Hanyong Chen a , Jae Young Kim b , Ann M. Bode a , Zigang Dong a,⁎ a

The Hormel Institute, University of Minnesota, 801, 16th AVE, NE, Austin, MN 55912, USA Kyungpook National University, Center for Laboratory Animal Resources, School of Animal BT Science, Department of Biochemistry, School of Dentistry, Dae-gu, Republic of Korea

b

Received 6 June 2013; received in revised form 27 March 2014; accepted 1 April 2014 Available online 13 April 2014

Abstract Nanog regulates human and mouse embryonic stem (ES) cell self-renewal activity. Activation of ERKs signaling negatively regulates ES cell self-renewal and induces differentiation, but the mechanisms are not understood. We found that ERK1 binds and phosphorylates Nanog. Activation of MEK/ERKs signaling and phosphorylation of Nanog inhibit Nanog transactivation, inducing ES cell differentiation. Conversely, suppression of MEK/ERKs signaling enhances Nanog transactivation to inhibit ES cell differentiation. We observed that phosphorylation of Nanog by ERK1 decreases Nanog stability through ubiquitination-mediated protein degradation. Further, we found that this phosphorylation induces binding of FBXW8 with Nanog to reduce Nanog protein stability. Overall, our results demonstrated that ERKs-mediated Nanog phosphorylation plays an important role in self-renewal of ES cells through FBXW8-mediated Nanog protein stability. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Introduction Embryonic pluripotent cells can give rise to differentiated progeny representing all three embryonic germ layers (Pera et al., 2000). Thus, embryonic stem (ES) cells are special

⁎ Corresponding author. Fax: + 1 507 437 9606. E-mail address: [email protected] (Z. Dong). 1 SH Kim and MO Kim contributed equally to this work. 2 Current Address: Department of Pharmacology, College of Pharmacy, The Catholic University of Korea, Bucheon 420-102, Republic of Korea.

primal structures in the body that retain two distinctive properties: the ability to self-renew through mitotic cell division to remain in an undifferentiated state and the ability to differentiate into a specific cell type (Patel and Yang, 2010). Stem cell factors, including Oct3/4, Sox2, Klf4, Nanog, c-Myc and Lin28, are crucial transcription factors known to regulate ES cells' self-renewal activity and/or to generate induced pluripotent stem (iPS) cells (Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Yu et al., 2007). Nanog occupies a central position in the transcriptional network of pluripotency (Boyer et al., 2005; Cole et al., 2008; Loh et al., 2006) and plays an essential role in early embryonic development (Mitsui et al., 2003). It is expressed in pluripotent

http://dx.doi.org/10.1016/j.scr.2014.04.001 1873-5061/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

2 embryonic cells, derivative ES cells, and the developing germ line of mammals and birds (Chambers et al., 2003; Lavial et al., 2007; Mitsui et al., 2003; Yamaguchi et al., 2005). Mouse Nanog-deficient inner cell mass (ICM) fails to generate epiblasts and only produces parietal endoderm-like cells (Mitsui et al., 2003). Conversely, ectopic expression of Nanog is sufficient to drive leukemia inhibitory factor (LIF; a key cytokine for maintenance of ES cell pluripotency)-independent self-renewal of undifferentiated ES cells (Chambers et al., 2003). Furthermore, 4 factors including Oct3/4, Sox2, Nanog and Lin28, are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit identical and essential characteristics of ES cells (Yu et al., 2007). Thus, Nanog plays a central role in ES cell self-renewal in developmental and de-differentiation processes. The maintenance of self-renewal and pluripotency in ES cells is controlled by extrinsic signaling pathways, intrinsic self-renewal factors and epigenetic modifications (Boiani and Scholer, 2005; Stewart et al., 2006). LIF, a member of the IL-6 cytokine family (Smith et al., 1988) provided by mitotic-inactivated feeder cells, stimulates ES cells through gp130-JAK-STAT3 signaling (Niwa et al., 1998), resulting in prevention of ES cell differentiation in the presence of serum (Matsuda et al., 1999). Besides the activation of STAT3, LIF also activates mitogen-activated protein (MAP) kinases, whose activation promotes differentiation of ES cells in mice (Burdon et al., 1999). Suppression of ERKs (extracellular-signal regulated kinases) signaling can promote ES cell self-renewal in mice (Burdon et al., 1999). Thus, mouse ES (mES) cells might maintain their self-renewal activity in the presence of LIF due to the balance of STAT3 activation and ERKs. Also, growth factors, such as LIF and bFGF, activate the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway (Jirmanova et al., 2002) by inhibiting glutathione synthase kinase 3 β (GSK3β), p53 and Nanog (Takahashi et al., 2003), as well as suppressing apoptosis in ES cells (Gross et al., 2005). In contrast to LIF-STAT3 signaling, activation of the Wnt pathway by 6-bromoindirubin-3-oxime (BIO), a specific inhibitor of GSK3, maintains the undifferentiated phenotype in ES cells and sustains expression of ES cell specific markers (Sato et al., 2004) through a synergistic effect with LIF-STAT3 signaling (Hao et al., 2006). Transforming growth factor beta (TGFβ)/ activin/nodal signaling plays an important role in ES cell self-renewal. Gene expression analysis indicates that TGFβ and its correlate factors, including Nodal Cripto Lefty1 and Lefty 2, are expressed at high levels in undifferentiated human ES cells (Sato et al., 2003). The TGFβ/activin/nodal pathway is activated through the Smad2/3 transcription factors in undifferentiated cells and Smad2/3 activation suppresses Smad1/5 activity, suggesting that TGFβ/activin/ nodal signaling can negatively regulate bone morphogenetic protein 4 (BMP4) in human ES cells (Beattie et al., 2005; James et al., 2005). However, the mechanisms are not understood. The ubiquitin–proteasome degradation mechanism is associated with most intracellular proteins, including membranesurface receptors (Gesbert et al., 2005; Martinez-Moczygemba et al., 2007; Rocca et al., 2001; Wauman et al., 2011). Over 60 Fbox proteins have been identified, but only a few, such as β-TrCP and FBXW7, are well characterized. Fbox proteins bind in the substrate-binding motif used for recognition and interaction with phosphorylated substrates (Kim et al., 2012; Skowyra et al., 1997).

S.-H. Kim et al. Recent studies indicate that post-translational modifications of iPS factors regulate their activity. For example, phosphorylation of human Sox2 at Ser249, Ser250 and Ser 251 (Van Hoof et al., 2009) inhibits Sox2 DNA binding activity (Tsuruzoe et al., 2006). Acetylation of mouse Sox2 at Lys75 by p300/CBP enhances nuclear export and degradation of Sox2 through an ubiquitin-mediated degradation pathway (Baltus et al., 2009). Furthermore, phosphorylation of human Oct4 at Ser229 by PKA might partially regulate Oct4 transactivation (Baltus et al., 2009). Phosphorylation of Nanog is associated with Pin1 (Moretto-Zita et al., 2010). These findings indicate that post-translational modifications of iPS factors are likely involved in the regulation of their activity, which could result in modulation of ES cell selfrenewal activity. Herein, we identified mechanisms for the self-renewal of ES cells showing that regulation of Nanog protein stability occurs through ERK1-mediated phosphorylation and ubiquitination.

Materials and methods Reagents and antibodies Dulbecco Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) were from Invitrogen (Carlsbad, CA). Restriction enzymes were obtained from New England BioLabs, Inc. (Ipswich, MA). The DNA ligation kit (v2.0) was from TaKaRa Bio, Inc. (Otsu, Shiga, Japan). Lipofectamine 2000 transfection reagents were obtained from Invitrogen and the JetPEI transfection reagent for 293 cells was from Q-Biogene (West Chester, PA). The pcDNA3.1 plasmid was purchased from Life Technologies, Inc. (Carlsbad, CA). The luciferase assay substrate was acquired from Promega (Madison, WI). The monoclonal-HA, His or Flag antibodies were from SigmaAldrich (St. Louis, MO) and Invitrogen, respectively. Antibodies against Nanog, phosphorylated ERKs (Thr202/Tyr204) and total ERKs were from Abcam (Cambridge, MA) and Cell Signaling Technology, Inc. (Danvers, MA), respectively. The antibody to detect phosphorylated Nanog (Ser52) was produced by Biosynthesis Inc. (Lewisville, TX).

Cell culture HEK293 and HeLa cells were cultured in DMEM and 10% heat-inactivated FBS in a 37 °C, 5% CO2 incubator. Cells were maintained by medium change every other day and then split at 90% confluence. Mouse ES cells (E14Tg2a) were maintained on CF-1 feeder cells, which were treated with mitomycin-C in standard complete ES cell culture medium supplemented with 15% heat-inactivated ES qualified FBS (Millipore, Billerica, MA), 0.055 mM β-mercaptoethanol (Invitrogen), 2 mM L-glutamine, 0.1 mM MEM non-essential amino acids and 1000 U/mL leukemia inhibitory factor (LIF; Millipore, Billerica, MA). ES cells were passaged at 70% confluence on the feeder cells.

Construction of expression vectors For the mammalian two-hybrid system assay and overexpression of Nanog, the open reading frame of mouse Nanog

ERK1 phosphorylates Nanog to regulate protein stability was amplified and introduced into the pACT mammalian two-hybrid system vector or pCMV-HA expression vector. For the GST-fusion protein of Nanog, full length Nanog (Nanog-1-305) was constructed by PCR-based amplification and introduced into the pGEX-5X-C vector (Cho et al., 2007). All constructs were confirmed by restriction enzyme mapping and DNA sequencing.

Western blotting Cells were harvested and disrupted with NP-40 lysis buffer [150 mM NaCl, 50 mM Tris–HCl pH 7.5, 1% Nonidet P-40, and Complete Protease Inhibitor Cocktail Tablets (Roche, Indianapolis, IN)] with thawing and freezing. The proteins were resolved by SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes, and target proteins were visualized using an enhanced chemiluminescence (ECL) detection kit with specific antibodies.

Mammalian two-hybrid assay The mammalian two-hybrid (M2H) assay was conducted according to the manufacturer's instructions (Checkmate Mammalian Two-Hybrid System assay; Promega). Briefly, HEK293 cells were maintained in 10% FBS-DMEM, seeded (2.0 × 104) into 48-well plates, and cultured 18 h before transfection. Various plasmids, including pACT-Nanog, pBIND-kinases and pG5-luciferase were combined in the same molar ratio (1:1:1) and the total amount of DNA was not more than 100 ng/well. The transfections were performed using jetPEI following the manufacturer's protocols. For the luciferase assay, the cells were disrupted by the addition of luciferase lysis buffer and incubated for 30 min at room temperature with gentle shaking. The luciferase activity was measured by a computer-controlled luminometer (Luminoskan Ascent, MTX Lab, Inc., McLean, VA) with the automatic addition of 100 μL substrate buffer. The relative firefly luciferase activity was calculated using the pG5-luciferase basal control and normalized against Renilla luciferase activity, which included the pBIND vector as an internal control.

Immunoprecipitation assay The wildtype pCMV-HA-Nanog, pcDNA4-His-ERK1, and pcDNA4His-ERK2 plasmids were transfected (as indicated) into HEK293 cells and cultured 36 h. The cells were harvested and disrupted with NP-40 lysis buffer. Protein binding was confirmed by immunoprecipitation and Western blotting with specific primary and HRP-conjugated secondary antibodies as indicated using an enhanced chemiluminescence (ECL) detection kit.

In vitro kinase assay Wildtype GST-Nanog, truncated deletion or point mutant proteins were used for an in vitro kinase assay with active ERK1 or ERK2 (Millipore). Reactions were carried out at 30 °C for 30 min in a mixture containing 50 μmol/L unlabeled ATP and 10 μCi of [γ-32P] ATP. The reaction was stopped by adding 6 × SDS sample buffer and samples were boiled and separated by 10% SDS-PAGE and visualized by autoradiography.

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Nanog activity assay To examine transactivation of Nanog, E14Tg2a ES cells (2.0 × 104) were seeded into 48-well plates and cultured with complete ES medium containing LIF for 18 h before transfection. The p5xGal4-luciferase and pBIND-Nanog wildtype, pmu6 pro si-ERK1, or pCDNA4-ERK1 plasmids were transfected with various combinations of expression vectors as indicated and cells were cultured for 36 h. The cells were disrupted with lysis buffer and luciferase activity was measured. The relative firefly luciferase activity was normalized against Renilla luciferase activity, which was obtained by co-transfection of the Renilla luciferase reporter plasmid (phRL-SV40).

Alkaline phosphatase staining To stain for colonies, ES cells were cultured in the appropriate medium, fixed with 2% formalin for 2 min at room temperature, and then alkaline phosphatase (AP) staining was performed with the Alkaline Phosphatase Detection Kit (Millipore) following the manufacturer's protocols. AP-positive colonies were observed under a light microscope (Olympus, Center Valley, PA).

RT-PCR and real time Total RNA was extracted with Trizol reagent (Invitrogen). One microgram of total RNA was used for the reverse transcription reaction with the SuperScriptII RT-PCR kit (Gibco BRL) following the manufacturer's recommendations. PCR was performed with ExTaq (TaKaRa, Japan). Gene expression of REX-1, Brachury, FGF5, Sox2, Oct4, and Nanog was analyzed by polymerase chain reaction (PCR) with gene specific primers (Supplemental Table 1). Gene expression of Nanog, Oct4, Sox2, and Brachyury was also analyzed by real time PCR (Supplemental Table 2).

Protein stability and ubiquitination To examine Nanog protein stability, E14Tg2a ES cells were cultured and treated with MG132 and cyclohexamide (CHX). Cells were harvested and protein levels were visualized by Western blotting with specific antibodies. Protein levels were determined at 2, 4, and 6 h after CHX treatment. To assess ubiquitination, the pcDNA3-Flag-Ub expression vector was co-transfected into E14Tg2a ES with combinations of wildtype and mutant Nanog and CA-MEK1, as indicated. Cells were cultured for 36 h and then proteins were extracted. Ubiquitination of Nanog proteins was visualized by immunoprecipitation with a Flag or HA antibody and Western blotting as indicated using an enhanced chemiluminescence (ECL) detection kit.

Results Nanog is a novel substrate of ERK1 Stem cell factors, including Sox2, Nanog, Oct3/4, Myc and Klf4, play a key role in self-renewal of embryonic stem (ES)

4 cells and reprogramming to induced pluripotent stem (iPS) cells (Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Yu et al., 2007). Recent studies indicate that posttranslational modifications, such as phosphorylation, acetylation, ubiquitination and sumoylation of Sox2 and Oct4 modulate their transactivation and/or transcriptional activity (Baltus et al., 2009; Saxe et al., 2009; Tsuruzoe et al., 2006; Van Hoof et al., 2009). Thus, we hypothesized that post-translational modifications, especially phosphorylation, of Nanog could modulate its activity. To identify potential kinase(s) of Nanog, we conducted a mammalian two-hybrid system assay (Cho et al., 2005) and found that ERK1 showed the highest binding activity with Nanog (Fig. 1A). To determine whether Nanog is a selective substrate of ERK1 or ERK2, we conducted an in vitro kinase assay with [γ-32P] ATP, active ERK1 or ERK2, and GST-Nanog, which was constructed and purified in our laboratory. The RSK2 N-terminal was used as a positive substrate control for ERK1 and 2. Autoradiography results indicated that ERK1 strongly phosphorylated Nanog and ERK2 showed only a weak phosphorylated band even though ERK1 and ERK2 were used at identical enzyme units of activity (Fig. 1B). To determine whether serines 52, 65, 71, and 78 are the target amino acids of Nanog that are phosphorylated by ERK1, we constructed a point mutant (serine to alanine) of each in GST-Nanog-1-305 and then purified the proteins and conducted an in vitro kinase assay with [γ-32P] ATP and active ERK1. We found that the point mutants S52A, S71A and S78A, but not S65A, exhibited substantially decreased 32P-labeled band density (Fig. 1C). Furthermore, we found that the triple mutation of Nanog, including Ser52, exhibited the most substantially decreased 32 P labeled band density (Fig. 1D, lanes 3–5 vs. 2). These results indicated that activated ERK1 phosphorylates serines 52, 71, and 78, but not 65, on Nanog. Using ectopic expression and co-immunoprecipitation, we found that ERK1 or 2 and Nanog interacted in an ex vivo cell culture system (Fig. 1E).

S.-H. Kim et al. Overall, these results demonstrated that Nanog is a novel binding partner and substrate of ERK1.

ERK1 signaling negatively regulates Nanog transactivation Stem cell factors, including Nanog, are transcription factors that regulate gene expression of downstream target genes involved in stem cell self-renewal and iPS production (Chambers et al., 2003; Mitsui et al., 2003). To examine the effect of ERK1 phosphorylation on the transactivation activity of Nanog, we constructed a pGal4-Nanog expression vector and analyzed the transactivation activity of Nanog using a p5xGal4-luciferase reporter assay and knocking down ERK1 expression. Knockdown of ERK1 increased Nanog transactivation activity in a dose-dependent manner corresponding with the dose-dependent decrease in the ectopic expression level of ERK (Fig. 1F). Furthermore, the transactivation activity of Nanog was decreased dose-dependently by co-expression of ERK1 (Fig. 1G). These result indicated that phosphorylation of Nanog negatively regulates Nanog transactivation activity, suggesting that the ERK1-Nanog signaling axis might negatively regulate ES cell self-renewal activity.

ERK1-Nanog signaling modulates self-renewal activity of ES cells Recent studies indicated that the MAP kinase signaling pathway plays an important role in the homeostasis of stem cell self-renewal activity (Ying et al., 2008). To examine the relationship between Nanog and ERKs activation in the differentiation process, we induced differentiation of ES cells by withdrawal of LIF and confirmed the protein level of Nanog and phosphorylation level of ERKs. The results indicated that the Nanog protein level was decreased and ERKs

Figure 1 Nanog is a novel substrate of ERK1 and ERK1 signaling negatively regulates Nanog transactivation activity. A, mammalian two-hybrid assay assessment of the ex vivo protein–protein interactions of pACT-Nanog and various pBIND–protein kinases. Activity is indicated by relative luminescence units normalized to a negative control (value for cells transfected with only pG5-luciferase [luc]/ pACT-Nanog = 1.0). Data are shown as means ± S.D. of values obtained from triplicate experiments. Significant differences were evaluated using the Student's t-test (*, p b 0.05, **, p b 0.005). B, Nanog is phosphorylated by ERK1 and ERK2. To confirm the phosphorylation of Nanog by ERK1 or ERK2, a GST-Nanog-1-305 fusion protein was purified and directly subjected to an in vitro kinase assay with [γ-32P] ATP and active ERK1 or ERK2, and then visualized by autoradiography. Each asterisk indicates the respective GST fusion protein. C, Confirmation of Nanog phosphorylation by ERK1. The GST-Nanog-1-305-S52A, -Nanog-1-305-S65A, -Nanog-1-305-S71A and -Nanog-1-305-S78A proteins were purified and subjected to an in vitro kinase assay with [γ-32P] ATP and active ERK1. The phosphorylated bands were visualized by autoradiography. D, The GST-Nanog-1-96-S52, 65, 71A, -Nanog-1-96-S52, 65, 78A, -Nanog-1-96-S52, 71, 78A, -Nanog-1-96-S65, 71, 78A and -Nanog-1-96-S52, 65, 71, 78A fusion proteins were purified and subjected to an in vitro kinase assay with [γ-32P] ATP and active ERK1. The phosphorylated bands were visualized by autoradiography. E, confirmation of Nanog and ERK1 and ERK2 binding. To confirm the binding of Nanog with ERK1 and/or ERK2, the pCMV-HA-Nanog-1-305 construct was co-transfected with pcDNA4-HisG-ERK1 into HEK293 cells. After culturing for 36 h, cells were disrupted with NP-40 lysis buffer and immunoprecipitated with a HisG monoclonal antibody. Co-immunoprecipitated Nanog was detected by Western blot (WB) using an HRP-conjugated HA antibody. F, knockdown of ERK1 induces Nanog transactivation activity. The p5xGal4-luciferase reporter plasmid and pGal4-Nanog-1-305-WT plasmid, which also harbors the Renilla luciferase reporter gene, were co-transfected with different doses of pmU6si-ERK1 into E14 cells and cultured 36 h. The luciferase activity was measured and normalized against Renilla luciferase activity. Data are shown as means ± S.D. of values obtained from triplicate experiments and significant differences were evaluated using the Student's t-test (*, p b 0.005). G, ectopic expression of ERK1 suppresses Nanog transactivation activity. The p5xGal4-luciferase reporter and pGal4-Nanog-1-305-WT plasmids were co-transfected with different doses of pCDNA4-HisG-ERK1 and cultured for 36 h. The luciferase activity was measured and normalized against Renilla luciferase activity. Data are shown as means ± S.D. of values obtained from triplicate experiments and significant differences were evaluated using the Student's t-test (*, p b 0.005).

ERK1 phosphorylates Nanog to regulate protein stability

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6 phosphorylation increased in a time-dependent manner (Fig. 2A). Additionally, to confirm the localization of Nanog and phosphorylated ERKs, we induced ES cell differentiation and treated cells with PD98059 to inhibit ERKs phosphorylation. The Nanog protein is always localized in both the nucleus and cytoplasm. Phosphorylated ERKs is also expressed in both the nucleus and cytoplasm, but translocates to the nucleus where it is concentrated during ES cell differentiation. However, PD98059 prevents ERKs phosphorylation and increases Nanog protein level (Supplemental Fig. 1A, B). To explore ERK1-Nanog signaling in ES cells, we determined the optimum concentration of LIF needed to obtain the maximum difference for studying the inhibitory effect of ERKs signaling induced by PD98059, a MEKs inhibitor. Results indicated that 10 units of LIF allowed the most gradual differentiation during the 4 day-differentiation process (Kim et al., 2012). Therefore, we co-treated ES cells with LIF (10 IU) and various doses of PD98059 (0, 5, 10, 20 μM). We found that inhibition of ERKs signaling increased the number of AP-positive colonies in a dose-dependent manner (Fig. 2B). Differentiation markers (i.e., Brachury and FGF5) were substantially suppressed by treatment with PD98059 dose-dependently under

S.-H. Kim et al. LIF-containing conditions (Fig. 2C). Furthermore, nondifferentiation markers, including REX-1, were increased substantially (Fig. 2C). Transcription of REX-1 is regulated by Nanog (Shi et al., 2006). Under LIF-withdrawal conditions, brachery was decreased and Sox2 protein levels were increased by treatment with PD98059 without changes in FGF5 or REX-1 (Fig. 2C). We treated ES cells with LIF (10 IU) and PD98059 (0, 5, 10, 20 μM) and found that ERKs phosphorylation was inhibited by increasing the amount of PD98059 in complete ES culture medium without altering the total ERKs protein level. The total protein level of Nanog was increased dose-dependently by PD98059, suggesting that ERKs might have a role in stabilizing Nanog (Fig. 2D). These results indicated that the ERK1-Nanog signaling axis negatively regulates self-renewal activities during ES cell maintenance.

Nanog phosphorylation plays an important role in protein stability Previous results indicated that the Nanog protein level was decreased and phosphorylated ERKs were increased in the

Figure 2 ERK1-mediated Nanog phosphorylation plays a critical role in ES cell self-renewal. A, protein levels of Nanog and ERKs during ES cell differentiation. E14Tg2a ES cells were forced to differentiate by withdrawal of LIF and the proteins were extracted at the indicated time point. The protein levels of Nanog, FBXW8, and phosphorylated and total ERKs were analyzed by Western blotting. β-Actin was used as an internal control to verify equal protein loading. B, inhibition of ERKs signaling by PD98059 suppresses ES cell differentiation. E14Tg2a ES cells were cultured for 4 days in LIF-reduced ES medium containing different doses of PD98059. The cells were fixed and stained using an alkaline phosphatase (AP) staining kit. The colonies were observed and AP-positive colonies were counted under a light microscope (× 40). C, inhibition of ERKs signaling restores ES cell differentiation markers. E14Tg2a ES cells were cultured for 4 days in LIF-reduced ES medium containing different doses of PD98059. Gene expression of non-differentiation markers including REX-1, Sox2 and differentiation markers, including brachyury and FGF5, was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) with gene specific primers. GAPDH was used as an internal control to verify equal amounts of cDNA. D, inhibition of ERKs signaling restores Nanog protein level. E14Tg2a ES cells were cultured for 4 days in LIF-reduced ES medium containing different doses of PD98059. The proteins were extracted and visualized by Western blotting with specific antibodies. β-Actin was used as an internal control to verify equal protein loading.

ERK1 phosphorylates Nanog to regulate protein stability

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Figure 3 Nanog is a novel substrate of FBXW8 and modulates protein degradation through the ubiquitin mediated proteosomal degradation pathway. A, the Nanog protein level is regulated through the proteosomal degradation pathway. E14Tg2a ES cells were treated with cyclohexamide (10 μg/mL) with or without MG132 (10 μM), an inhibitor of the proteosomal degradation pathway, and then proteins were extracted at the indicated time point. The Nanog protein level was visualized by Western blotting using a Nanog specific antibody. β-Actin was used as an internal control to verify equal protein loading. B, PD98059 inhibits ubiquitination of exogenous Nanog in ES cells. The indicated combination of expression vectors was co-transfected into E14Tg2a ES cells and cultured for 36 h in LIF-deficient ES medium. The ubiquitination of Nanog was visualized by immunoprecipitation with anti-HA and Western blotting with anti-Ub. C, endogenous ubiquitination of Nanog during ES cell differentiation depends on phosphorylated ERK. PD98059 inhibits ubiquitination of endogenous Nanog in ES cells. The ubiquitination of Nanog was visualized by immunoprecipitation with anti-Nanog and Western blotting with anti-Ub. D, the pcDNA3-Flag-βTrCP1, pcDNA3-Flag-βTrCP2, pcDNA3-Flag-FBXW2, pcDNA3-Flag-FBXW4, pcDNA3-Flag-FBXW5, pcDNA3-Flag FBXW7α, and pcDNA3-Flag-FBXW8 plasmids were each co-transfected with pCMV-HANanog-1-305-WT into HEK293 cells and cells cultured for 36 h. The proteins were extracted with NP-40 cell lysis buffer. The Nanog proteins were immunoprecipitated with anti-HA and the co-immunoprecipitated FBXW4, FBXW5 and FBXW8 proteins were visualized by Western blotting with anti-Flag. The ectopic protein levels of the Flag-Fbox proteins and HA-Nanog were visualized by Western blotting with specific antibodies using the same cell lysate. β-Actin was used as an internal control to verify equal protein loading. E, Nanog mutations at serines 52, 65, 71 and 78 abrogate Fbox protein binding. Combinations of expression vectors, including HA-Nanog-WT, HA-Nanog-mt, Flag-FBXW4, Flag-FBXW5 and Flag-FBXW8 as indicated, were co-transfected into HEK293 cells and cultured for 36 h. The proteins were immunoprecipitated with anti-HA and the co-immunoprecipitated Fbox proteins were visualized by Western blotting using anti-Flag. The ectopic protein levels of Flag-FBXW4, Flag-FBXW5 and Flag-FBXW8 and HA-Nanog were visualized by Western blotting with specific antibodies using the same cell lysate. β-Actin was used as an internal control to verify equal protein loading.

8 differentiation process by withdrawal of LIF (Fig. 2A). These results suggest that the stability of the Nanog protein might play an important role in the regulation of its activity. To address this hypothesis, we analyzed protein stability by treatment with MG132, a proteosomal inhibitor, in either the presence or absence of cyclohexamide (CHX), an inhibitor of translation, in ES cells after withdrawal of LIF. We found that CHX treatment rapidly reduced the Nanog protein level and MG132 treatment restored the Nanog protein level (Fig. 3A). Importantly, we further found that the MEK inhibitor, PD98059, reduced the ubiquitination of Nanog (Fig. 3B). Endogenous ubiquitination of Nanog was increased during ES cell differentiation and phosphorylated ERKs also increased. Inhibition of ERKs by PD98059 decreased the ubiquitination of Nanog (Fig. 3C). These results clearly demonstrated that the Nanog protein level is regulated by protein stability through ERKs phosphorylation and phosphorylation of Nanog plays a critical role in the regulation of Nanog protein stability.

FBXW8 is responsible for Nanog stability The Fbox protein is the substrate-recognition component among the families of E3 ubiquitin ligases (Nandi et al., 2006). To identify the E3 ligase responsible for targeting Nanog, we used immunoprecipitation to screen several Fbox E3 ligases including β-TrCP1 (FBXW1), β-TrCP2 (FBXW11), FBXW2, FBXW4, FBXW5, FBXW7α and FBXW8 (Fig. 3D). Notably, we found that FBXW4, FBXW5 and FBXW8 were

S.-H. Kim et al. co-immunoprecipitated with Nanog (Fig. 3D, lanes 6, 7 and 9). Very importantly, strong binding of wildtype Nanog with FBXW8 induced by CA-MEK1 was totally abrogated in the mutant Nanog (Fig. 3E). These results indicated that the FBXW8 is the most responsible partner for Nanog stability.

FBXW8 regulates Nanog stability To confirm the role of FBXW8, we introduced knockdown (Supplemental Fig. 2) and overexpression systems (Supplemental Fig. 3A, B). Exogenous transcription was dramatically increased by ectopic expression of FBXW8 and the total level of FBXW8 transcription was slightly increased (Supplemental Fig. 3A). Quantitative real time RT-PCR results showed that endogenous transcription was not changed (Supplemental Fig. 3B). Knockdown or overexpression of FBXW8 has an effect on ES cell differentiation. Quantitative real time RT-PCR results confirmed the effect of FBXW8 on ES cell markers (Supplemental Fig. 4). Transduction with shFBXW8 substantially extended the half-life of endogenous Nanog (Fig. 4A) and increased protein stability in ES cell differentiation (Fig. 4B). Further, FBXW8 overexpression shortened the half-life of endogenous Nanog (Fig. 4C) and alkaline phosphatase assay results indicated that knockdown of FBXW8 delayed ES cell differentiation (Fig. 4D). These results demonstrated that FBXW8 is an E3 ligase that is responsible for reducing Nanog protein stability, resulting in ES cell differentiation.

Figure 4 Function of FBXW8 in ES cells. A, E14 cells were introduced with lentivirus containing pLK0.1-Mock or pLK0.1-shFBXW8. The half-life of endogenous Nanog in control or FBXW8 knockdown cells was assessed using the cyclohexamide (CHX) chase assay. B, ES cells were introduced with lentivirus containing pLK0.1-Mock, and pLK0.1-shFBXW8. The protein stability of endogenous Nanog was assessed in control or FBXW8 knockdown cells cultured without LIF in ES medium for the indicated days. C, ES cells were introduced with pCDNA3-Flag-FBXW8 and the half-life of endogenous Nanog is shown by Western blot. D, knocking down FBXW8 expression enhances stem cell self-renewal activity. The pLK0.1-mock or pLK0. 1-FBXW8 lentivirus was introduced into E14Tg2a cells and then cells were cultured without LIF in ES medium for 3 days, fixed and stained using an AP staining kit. The AP-positive colonies were counted under a light microscope (× 40). Data are shown as means ± S.D. of values obtained from triplicate experiments. Significant differences were evaluated using the Student's t-test (*, p b 0.05).

ERK1 phosphorylates Nanog to regulate protein stability

Discussion Although the methylation status of gene promoters of stem cell factors is believed to be a major regulatory mechanism to govern stem cell self-renewal activity, recent studies have demonstrated that post-translational modifications, including phosphorylation, sumoylation and ubiquitination, of stem cell factors, such as Sox2 and Oct4, might play an important role in the regulation of transcriptional activity (Baltus et al., 2009; Saxe et al., 2009; Tsuruzoe et al., 2006; Van Hoof et al., 2009). Recent studies also indicate that Nanog, an ES cell-specific homeodomain protein, is required for the self-renewal of ES cells (Chambers et al., 2003; Mitsui et al., 2003). Self-renewal and maintenance of pluripotency of mouse ES cells require leukemia inhibitory factor (LIF; a member of the IL-6 cytokine family), which is used for maintenance of ES cells in an undifferentiated state (Smith et al., 1988; Williams et al., 1988). At the same time, LIF also stimulates and activates the MAP kinase pathway, the activation of which induces differentiation of ES cells. However, suppression of ERKs signaling promotes the self-renewal activity of mES cells (Burdon et al., 1999). Therefore, the ERKs signaling pathway is believed to play an important role in the regulation of ES cell self-renewal activity. In this study, we found that ERK1, but not ERK2, strongly binds with and phosphorylates Nanog in vitro (Fig. 1A, B, E). This finding is important because ERKs signaling induces ES cell differentiation (Burdon et al., 1999) and the MEK inhibitor, PD0325901, enhances ES cell self-renewal activity (Ying et al., 2008). Furthermore, our results demonstrated that ERK1 signaling negatively modulates Nanog transactivation activity (Fig. 1F, G) and enhances ubiquitination of Nanog (Fig. 3B, C). The down-regulation of the protein level of Nanog during ES cell differentiation corresponded with increased levels of phosphorylated ERKs (Fig. 2A). Therefore, we concluded that the phosphorylation of Nanog mediated by ERK1 is critical for the down-regulation of Nanog through ubiquitinationmediated protein stability. In order to regulate the protein level through the proteosomal protein degradation pathway, the binding and interaction of the ubiquitin conjugation machinery with the target protein are necessary (Hochstrasser, 1996). However, the molecular mechanism explaining how the phosphorylation of Nanog affects stability is not yet clearly understood. Another group identified the same phosphorylation sites in the Nanog protein (22). However, they did not show a direct phosphorylation of Nanog but showed that it was phosphorylated at multiple Ser/Thr-Pro motifs by an unidentified kinase. We showed the direct phosphorylation of Nanog by ERK1 (Fig. 1B) and clearly showed that Ser52 is the major phosphorylation site and Ser65 is weakly phosphorylated by ERK1 (Fig. 1C, D). Using similar experimental conditions to study protein stability, we found that phosphorylation of Nanog by ERK1 resulted in decreased Nanog stability and inhibition of ERK1 increased stability. In contrast, others found that phosphorylation of multiple Ser/Thr-Pro motifs increased Nanog stability (22). This suggests that the exact phosphorylation site(s) and/or kinase involved can determine whether Nanog is stabilized or degraded. Many transcription factors are involved in ES cell self-renewal and the regulation of pluripotency. Nanog has been shown to exhibit auto-regulation (Loh et al., 2006).

9 Although Nanog is a key regulator for ES cell self-renewal (Ying et al., 2008), a direct mechanism for regulating Nanog activity has not been elucidated. Our findings demonstrated that ERK1 is a major direct effector kinase of Nanog (Fig. 1). We found that activation of ERK1 signaling inhibited transactivation activity of Nanog (Fig. 1G). In contrast, blocking ERK1 signaling by siRNA-ERK increased the transactivation activity of Nanog (Fig. 1F). The effects further affected ES cell self-renewal activity (Fig. 2B, C). These results correspond well with previous studies showing that the MEKs/ERKs signaling pathway negatively regulates ES cell self-renewal activity (Burdon et al., 1999). In addition, quantitative real time RT-PCR results showed that Sox2, but not Oct4, was affected by FBXW8 directly or indirectly. However, future studies are needed. A number of transcription factors are required for pluripotency of stem cells. Nanog, a homeobox transcription factor, plays a critical role in regulating embryonic stem cell pluripotency (Ramakrishna et al., 2011). Our study has highlighted a degradation mechanism of Nanog through FBXW8 as a recognition component (Fig. 3D, E). Nanog is a novel substrate of FBXW8 in proteolytic processing by ubiquitin-mediated degradation.

Summary Our findings demonstrate a molecular mechanism explaining how ERKs signaling negatively regulates Nanog activity in ES cells. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.scr.2014.04.001.

Acknowledgments This work was supported by the Hormel Foundation and Cooperative Research Program for the Next-Generation BioGreen 21 Program (Project No. PJ009560).

We thank Nicki Brickman at the Hormel Institute, University of Minnesota for assistance in submitting this manuscript.

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