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localization of the carbohydrate responsive binding protein (ChREBP) by two nuclear export signal sites: Discovery of a new leucine-rich nuclear export signal site
Biochemical and Biophysical Research Communications 391 (2010) 1166–1169
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Coordinate regulation/localization of the carbohydrate responsive binding protein (ChREBP) by two nuclear export signal sites: Discovery of a new leucine-rich nuclear export signal site Masashi Fukasawa a, Qing Ge a, R. Max Wynn a, Seiji Ishii a, Kosaku Uyeda a,b,* a b
Biochemistry Department, University of Texas Southwestern Medical Center, Dallas, TX 75390-9038, USA Dallas Veterans Affairs Medical Center, Dallas, TX 75216, USA
a r t i c l e
i n f o
Article history: Received 12 November 2009 Available online 17 December 2009 Keywords: Carbohydrate responsive element binding protein Nuclear import/export signal sites Lipogenesis Transcription factor Glucose signals and 14-3-3
a b s t r a c t Carbohydrate response element binding protein (ChREBP) is responsible for conversion of dietary carbohydrate to storage fat in liver by coordinating expression of the enzymes that channel glycolytic pyruvate into lipogenesis. The activation of ChREBP in response to high glucose is nuclear localization and transcription, and the inactivation of ChREBP under low glucose involves export from the nucleus to the cytosol. Here we report a new nuclear export signal site (‘‘NES1”) of ChREBP. Together these signals provide ChREBP with two NES sequences, both the previously reported NES2 and now the new NES1 coordinate to interact together with CRM1 (exportin) for nuclear export of the carbohydrate response element binding protein. Published by Elsevier Inc.
Introduction The liver functions as the principal organ responsible for the conversion of excess ingested carbohydrate, which is stored as fat. A pivotal transcription factor, termed the Carbohydrate response element binding protein (ChREBP) controls expression of the carbohydrate response element (ChoRE)-containing genes. These tightly controlled genes include liver pyruvate kinase (LPK) and all of the liver lipogenic enzymes [1]. In response to high glucose concentrations, the activation of the ChREBP activity occurs at two levels: translocation into the nucleus and transcriptional control [2]. ChREBP is a large transcription factor consisted of 865 amino acids with Mr = 96,500 kDa (Fig. 1A). It contains several functional domains including nuclear export signal site (NES), nuclear import signal sites (NLS), a DNA binding bHLH/Zip domain, and prolinerich regions implicated in protein–protein interactions including multiple phosphorylation sites [1,3]. Previous studies [4] have shown that the N-terminal region of ChREBP (residues 1–251) interacts directly with a dimeric form of the 14-3-3 protein, and Abbreviations: LPK, liver pyruvate kinase; ChREBP, carbohydrate response element binding protein; NES, nuclear export signal; NLS, nuclear localization signal; ChoRE, carbohydrate response element; CRM1, chromosome region maintenance 1; WT, wild-type. * Corresponding author. Address: Biochemistry Department, University of Texas Southwestern Medical Center, Dallas, TX 75390-9038, USA. Fax: +1 214 648 8856. E-mail address: [email protected] (K. Uyeda). 0006-291X/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.bbrc.2009.11.115
is responsible for sub-cellular cellular localization, while the C-terminal region of ChREBP forms a complex with a Max-like protein X (Mlx) to bind to various DNA sites, thereby activating the transcriptional activity [3,5]. Previous studies have reported a nuclear export signal site (NES), which is located at the amino acid residues (86–95) of ChREBP [6]. In this communication we report an identification of a new leucine-rich NES site located at the N-terminus of ChREBP (residues 5–15), and the effects of site-directed mutations of the site on the nuclear localization and transcription activities in hepatocytes and in vitro binding of ChREBP-14-3-3 complex to CRM1 (exportin). The results demonstrate that both NES sites are essential for the translocation of ChREBP from nucleus into cytoplasm. We propose to call the new site ‘‘NES 1” and change the previously identified ‘‘NES” site to ‘‘NES 2”. Materials and methods Chemicals. All the reagents were purchased from Sigma unless otherwise stated. The 14-3-3 antibodies (K-19) were purchased from Invitrogen (Carlesbad, CA). CRM1 IgG and Lipofectamine were products of Santa Cruz (Santas Cruz, CA). The expression vector for GST-tagged human CRM1 (hCRM1) was a gift of Dr. Y. M. Chook (Pharmacology, UT Southwestern Med. Cent., Dallas, TX). His-tagged 14-3-3b, GST-tagged human CRM1 (hCRM1) and GST-tagged rat CRM1 (rCRM1) were expressed in Escherichia coli and purified as described previously [4].
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Fig. 1. (A) A schematic representation of the functional domains and phosphorylation sites for cAMP-dependent protein kinase of ChREBP. The locations of the functional domains are shown: Nuclear export signal sites, NES1, NES2; nuclear localization signal site, NLS; proline-rich region, bHLH/ZIP, and ZIP-like domains. The locations of three phosphorylation sites of PKA are indicated as P1, P2, and P3. (B) Alignment of N-terminal region of ChREBP from rat, mouse and human. The NES and NLS sequences are boxed. Two NES regions contain the amino acid sequences fit consensus F–x2–3–F–x2–3–F–x–F (F, hydrophobic amino acids; x is any amino acid). Point mutations used in this study are also indicated above the original amino acids. P1 is PKA-phosphorylation site (Ser196), and an alpha helix (a2 Helix) is 14-3-3 binding site.
15 min at 22 °C and finally washed with PBS containing 50 mM glycine. The GFP-derived fluorescence was observed and analyzed using a confocal laser microscope. Typically three sets of about 100 fluorescent cells were counted and scored for sub-cellular localization [2]. Electrophoresis mobility-shift assay (EMSA). Gel mobility-shift assays were performed as previously described [7]. ChREBP antiserum (Novus) was added to the reaction mixtures for super-shift assays. Double-stranded oligonucleotides were prepared by mixing equal amounts of the complementary single-stranded DNAs in 50 mM NaCl, heating to 70 °C for 15 min, and cooling to 22 °C. The annealed oligonucleotides were labeled with 32P in the presence of [c32P]ATP (Amersham Pharmacia) and T4 polynucleotide kinase (New England Biolabs). Binding reactions were carried out in a reaction mixture containing 20 mM Hepes (pH 7.9), 50 mM KCl, 5 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF, 10% glycerol, 1.25 lg poly(dI-dC) (Amersham Pharmacia), and 10–25 lg nuclear extract. The reaction mixture was incubated at 22 °C for 30 min, and DNA–protein complexes were separated by electrophoresis in a non-denaturing 4.5% PAGE gel. Protein–protein interactions of ChREBP with 14-3-3 and CRM1 (Exportin). In vitro binding assays of interaction between ChREBP or mNES-ChREBPs, 14-3-3b and CRM1 were carried out as follows. FLAG-tagged ChREBP expressed in HEK293T and purified from cell lysate by incubating with beads bearing anti-FLAG antibodies as described before [4]. In the binding buffer, the FLAG-ChREBPbound beads were incubated with either homogeneous hCRM1 or rCRM1 (3 lg) and 14-3-3b (2 lg) in the reaction mixture (0.5 ml) containing 20 mM HEPES (pH 7.3), 110 mM KOAc, 2 mM MgCl2, 5 mM NaOAc, 0.5 mM EGTA, and 0.01% Nonidet P-40 for 1.5 h at 4 °C with gentle rocking. After washing the beads (3 times) with reaction buffer without serum albumin, proteins were eluted with SDS–PAGE sample buffer. Samples were then subjected to PAGE and immunoblot detection for CRM1, 14-3-3, and ChREBP. Results
Construction of plasmids. Construction of expression vectors for ChREBP, GFP-tagged ChREBP, and FLAG-tagged ChREBP were described previously [4]. Oligonucleotides used for site-directed mutagenesis were; mNES1, 50 -ATGGCGCGCGCGGCGGCGGATCTA TCCGTGAACGCGCAGGTCCCCGG-30 and 50 -TccggggacctgcgcgttcaCG GATAGATCCGCCGCCGCGCGCGCGCCAT-30 . Immunoblot analysis. Protein samples were separated on either on an 8.0 % or a 10.0% SDS–PAGE gel and blotted onto Trans-Blot nitrocellulose membrane (Bio-Rad). The trans-blots were briefly rinsed with Tris-buffered saline (TBS) buffer containing 0.1% Tween-20 (TBS-T) then incubated for 1 h at 22 °C in TBS-T containing 5% non-fat milk (Difco). The blocked membrane was incubated with primary antibodies for 1 h at 22 °C. Secondary antibody, horseradish peroxidase-conjugated anti-rabbit IgG (ZyMed) were used as positive signals, which were visualized by ECL western blotting detection system (Amersham). Primary hepatocyte culture, transfection, and luciferase reporter assay. Isolation of hepatocytes by collagenase treatment from rat liver, transfection with Luciferase reporter and sub-cellular localization were performed as described before [4]. Sub-cellular localization of ChREBP. To analyze the sub-cellular localization of ChREBP, primary hepatocytes were plated onto 35 mm glass-bottomed dish (MatTek) coated with type I collagen at a density of 5.0 105 per dish. Onto the hepatocyte-containing plates either wild-type or the mutated (3.2 lg) forms of GFP-ChREBP-expressing plasmids were transfected by Lipofectamine 2000 (see above). After 16 h transfection 27.5 mM glucose was added for the high glucose condition, and after 24 h the cells were washed once with PBS, fixed with PBS buffered with 4% formaldehyde for
Mutations at either NES1 or NES2 sites affect ChREBP binding to CRM1 A computer search revealed a new leucine-rich nuclear export signal (NES) site, termed ‘‘NES1”, located at the N-terminus of mNES1 WT
mNES1/2
mNES2
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14-3-3
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Fig. 2. CRM1 and endogenous 14-3-3 bound to FLAG-ChREBP and mutants of NES (NES1, NES2 and NES1/2). Wild-type FLAG-ChREBP, mutants of NES1, NES2 and NES1/2 of FLAG-ChREBP were expressed in HEK293 cells and the expressed ChREBP were bound to anti-FLAG antibodies beads. The CRM1 and 14-3-3 bound to the beads were eluted and analyzed by PAGE and subjected to Western blotting as described under the Materials and methods section.
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ChREBP (5LADLSVNLQVP15), in addition to the previously reported ‘‘NES” site (85TLTRLFECLSLA95) [3]. In order to determine the role of NES1, we have prepared a mutant of the NES1 site (mNES1) by substituting two leucine residues with alanines (L5A and L12A). For comparison, we have employed the previously described mNES2 [4]. The wild-type-ChREBP (WT) and the mutants of both NES sites of ChREBP were expressed in HEK293 cells, following expression cell extracts were incubated with homogeneous rat CRM1 (hCRM1) (Fig. 2). Wild-type (WT)-ChREBP bound hCRM1 as well as the endogenous 14-3-3 proteins were tested for binding under these conditions (Fig. 2). Both mNES1- and mNES2-ChREBP also bound to CRM1 and to the endogenous 14-3-3, but by much less amounts compared to that with the WT-ChREBP. However, mutation of both NES sites (NES1 and NES2) of ChREBP completely abolished the CRM1 binding to ChREBP. The same results were obtained with the rat CRM1 protein. These results indicate that both NES1 and NES2 sites are required for maximum binding of CRM1. It is also worth noting that the amounts of the 14-3-3 proteins bound to WT-ChREBP and the individual NES mutants (either NES1 or
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NES2) of ChREBP were similar to those of CRM1 binding alone, suggesting that CRM1 may interact with ChREBP-14-3-3 heterodimer. Furthermore, these results suggest that the HEK293 cells contain sufficient endogenous 14-3-3 in the cells to form a stable ternary complex. In the export of proteins usually Ran GTPase and GTP are required [8], but the addition of an active form of purified Ran Q69L-GTPase [8] and GTP to the binding reaction mixture had no effect. The effects of mNES1 and mNES2 on the nuclear localization and transcriptional activities of ChREBP In order to further elucidate the roles of NES1 for export of ChREBP from the nucleus to the cytoplasm, we have determined the sub-cellular localization of GFP-ChREBP, and all three NES mutants in primary hepatocytes using confocal microscopy. GFP-tagged WT-ChREBP localized exclusively in the cytosol under low glucose conditions, but under high glucose ChREBP migrated to the nucleus (Fig. 3A and B). However, GFP-mNES1ChREBP was localized to the nucleus, either under low or high
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Fig. 3. (A) Sub-cellular localization of full-length ChREBP and its mutants the NES or NLS in primary rat hepatocytes under low glucose and high glucose condition. Primary cultured rat hepatocytes were co-transfected with GFP-tagged ChREBPs. The cells were cultured in D-MEM containing 5.5 mM glucose, and then incubated for additional 8 h with 5.5 mM (open bars) or 27.5 mM (filled bars) glucose-containing medium. Cells were fixed with 4% formaldehyde and localization of fluorescence was counted under confocal microscopy. The values presented are the mean and SD of three sets of about 100 fluorescent cells. mNES1, mNES2, mNES1/2 and mNLS mutations were introduced within NES1, NES2, both of NES1 and NES2, and NLS, respectively. (B) Representative picture of sub-cellular localization of wild-type and the NES mutants of ChREBP used in (A) under low glucose condition. (C) The effect of mutation of the NES sites on glucose-responsive transcriptional activity. Primary cultured rat hepatocytes were co-transfected with the ChREBP expression vectors as described in Materials and methods and the cells were incubated for 12 h in low and high glucose as above. Luciferase reporter activities were measured and are shown as the firefly luciferase activity [1]. The values presented are the mean and SD of replicate (three to four) in cultures from a single determinations represent more than three individual experiments. Total amount of DNA for expression vector was compensated with empty vector, pcDNA3.1.
M. Fukasawa et al. / Biochemical and Biophysical Research Communications 391 (2010) 1166–1169
glucose conditions. A similar nuclear localization was observed with mNES2, confirming our previous results [4]. GFP-mNES1/ 2-ChREBP also remained in the nucleus. However, unlike the NES mutants, a mutant of the nuclear localization signal site (NLS) mNLS-ChREBP was unable to localize ChREBP to the nucleus, as expected (Fig. 3A). These results demonstrated the importance of both NES1 and NES2 sites for export of ChREBP out of nucleus into the cytosol, because unlike WT-ChREBP these NES site mutants were unable to exit the nucleus even under low glucose conditions. The transcriptional activity assay of ChREBP in which the ChREBP-activated LPK promoter drove the expression of the luciferase reporter [1,4] revealed that WT-ChREBP was activated 3-fold greater under high glucose conditions as compared to low glucose conditions (Fig. 3C). However, mNES1-ChREBP showed approximately 36% lower activity in high glucose when compared to that of the WT-ChREBP and NES2 and NES1/2 both failed to show any transcriptional activity. In contrast to the nuclear localization activities, the differences in the transcriptional activities of the mNES1-ChREBP under high and low glucose (‘‘glucose sensitivity”) was retained, but the glucose activation was decreased approximately 30% compared to the WT-ChREBP. The transcriptional activities of the other mutants of the mNES2-ChREBP and that mNES1/2-ChREBP were completely lost. These results suggest that either the sub-cellular distribution assay is less sensitive to glucose concentration than the transcriptional activity or that some of the NES1 mutant remained in the nucleus, thereby retaining the transcriptional activity. Although the NES1/2-ChREBP localized in the nucleus, all transcriptional activity was lost. Further examination of this unexpected loss of the transcription activity of the ChREBP double mutant in spite of nuclear localization, revealed that the mNES1/2-ChREBP possessed DNA binding activity as demonstrated by the super-shift assay. The mutation of both NES sites showed additive effects on the nuclear localization of GFP-tagged ChREBP. NES1 and perhaps NES2 sites may play a role in glucose sensing since they both inactivated nuclear export and decreased glucose sensitivity. The export of CRM1 usually binds its cargo protein in the presence of Ran GTPase and GTP in the export [9]. However, Ran GTPase is not required for CRM1 binding of ChREBP as is in CRM1-SNUPN complex [10]. Discussion In response to increased circulating glucose, ChREBP is shifted from the cytoplasm to the nucleus, thereby down-regulating LPK expression and controlling lipogenic genes, which regulate fat storage. Under low glucose conditions such as starvation the transcriptional activity of ChREBP is terminated probably by phosphorylation mediated by protein kinases such as PKA, followed by interaction with 14-3-3, finally the ChREBP-14-3-3 complex binds to CRM1 to localize the ternary complex in the cytosol. Here we present a new export signal site of ChREBP termed ‘‘NES1” in addition to the previously reported ‘‘NES” site [6], now renamed ‘‘NES2”. The NES1 site (residues 5LADLSVNLQVP15) is located in the N-terminus of ChREBP. The evidence in support of this suggestion is based on the following results: the substitution of two Leu residues with Ala resulted in (a) the decreased CRM1
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binding in the in vitro ‘‘pull-down” assays, (b) a loss of the nuclear localization of GFP-tagged ChREBP mutants, and (c) a loss of transcriptional activity as determined using a reporter activity assay. It appears that both NES1 and NES2 sites are required for the maximum interaction with CRM1, and the maximum nuclear export and the transcriptional activities of ChREBP. Mutations in either the NES1 or the NES2 site alone affected ChREBP binding only partially. The amino acid sequnces of the nuclear export signal site (NES1) of ChREBP as well as NES2 do not seem to fit the consensus sequence of Ø–X(2–3)–Ø–X(2–3)–Ø–X–Ø (in which Ø is Lys, Val, Ile, Phe or Met, and X is any amino acid [10,11]. Moreover, recently the crystal structure of CRM1 bound to snurportin 1 (SNUPN) has been reported [10,12]. The amino acid sequence of ChREBP NES1 includes Met-1, Leu-5, Leu-8, Leu-12, and Val-14, which is nearly identical to that of SNUPN. According to the 3-D structure the N-terminal NES peptide of SNUPN is an a-helical-extended structure that binds to a highly conserved hydrophobic groove of CRM1 outer helices. Due to the sequence similarity, it is likely that the NES1 site in ChREBP possesses a combined helix-extended structure, which may also bind to the same NES-binding groove of CRM1. The second NES site of SNUPN is mainly basic and binds to an acidic surface of CRM1, which is situated adjacent to the first NES site. In contrast to the SNUPN, the NES2 epitope of ChREBP has one basic and one acidic residue, consequently it may not bind adjacent to NES1. However, the mutational effects on NES1 and NES2, in ChREBP, appear to be additive on all three biological activity assays performed here. This may favor both NES epitopes binding adjacent to each other, similar to the SNUPN example. Acknowledgments This work was supported by grants from the Department of Veterans Affairs and by National Institute of Health Grant DK063948. We thank Dr. Yuh M. Chook (Univ. Texas Southwestern Med. Cent., Dallas, TX) for the hCRM1 DNA and her valuable discussion. References [1] H. Yamashita, M. Takenoshita, M. Sakurai, R.K. Bruick, W.J. Henzel, W. Shillinglaw, et al., Proc. Natl. Acad. Sci. USA 98 (2001) 9116–9121. [2] T. Kawaguchi, M. Takenoshita, T. Kabashima, K. Uyeda, Proc. Natl. Acad. Sci. USA 98 (2001) 13710–13715. [3] S. Cairo, G. Merla, F. Urbinati, A. Ballabio, A. Reymond, Hum. Mol. Genet. 10 (2001) 617–627. [4] H. Sakiyama, R.M. Wynn, W.R. Lee, M. Fukasawa, H. Mizuguchi, K.H. Gardner, et al., J. Biol. Chem. 283 (2008) 24899–24908. [5] A.K. Stoeckman, L. Ma, H.C. Towle, J. Biol. Chem. 279 (2004) 15662–15669. [6] G. Merla, C. Howald, S.E. Antonarakis, A. Reymond, Hum. Mol. Genet. 13 (2004) 1505–1514. [7] S. Ishii, K. Iizuka, B.C. Miller, K. Uyeda, Proc. Natl. Acad. Sci. USA 101 (2004) 15597–15602. [8] D. Gorlich, U. Kutay, Annu. Rev. Cell. Dev. Biol. 15 (1999) 607–660. [9] D. Gorlich, N. Pante, U. Kutay, U. Aebi, F.R. Bischoff, EMBO J. 15 (1996) 5584– 5594. [10] X. Dong, A. Biswas, K.E. Suel, L.K. Jackson, R. Martinez, H. Gu, et al., Nature 458 (2009) 1136–1141. [11] U. Kutay, S. Guttinger, Trends Cell Biol. 15 (2005) 121–124. [12] T. Monecke, T. Guttler, P. Neumann, A. Dickmanns, D. Gorlich, R. Ficner, Science 324 (2009) 1087–1091.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Coordinate regulation/localization of the carbohydrate responsive binding protein (ChREBP) by two nuclear export signal sites: Discovery of a new leucine-rich nuclear export signal site Masashi Fukasawa a, Qing Ge a, R. Max Wynn a, Seiji Ishii a, Kosaku Uyeda a,b,* a b
Biochemistry Department, University of Texas Southwestern Medical Center, Dallas, TX 75390-9038, USA Dallas Veterans Affairs Medical Center, Dallas, TX 75216, USA
a r t i c l e
i n f o
Article history: Received 12 November 2009 Available online 17 December 2009 Keywords: Carbohydrate responsive element binding protein Nuclear import/export signal sites Lipogenesis Transcription factor Glucose signals and 14-3-3
a b s t r a c t Carbohydrate response element binding protein (ChREBP) is responsible for conversion of dietary carbohydrate to storage fat in liver by coordinating expression of the enzymes that channel glycolytic pyruvate into lipogenesis. The activation of ChREBP in response to high glucose is nuclear localization and transcription, and the inactivation of ChREBP under low glucose involves export from the nucleus to the cytosol. Here we report a new nuclear export signal site (‘‘NES1”) of ChREBP. Together these signals provide ChREBP with two NES sequences, both the previously reported NES2 and now the new NES1 coordinate to interact together with CRM1 (exportin) for nuclear export of the carbohydrate response element binding protein. Published by Elsevier Inc.
Introduction The liver functions as the principal organ responsible for the conversion of excess ingested carbohydrate, which is stored as fat. A pivotal transcription factor, termed the Carbohydrate response element binding protein (ChREBP) controls expression of the carbohydrate response element (ChoRE)-containing genes. These tightly controlled genes include liver pyruvate kinase (LPK) and all of the liver lipogenic enzymes [1]. In response to high glucose concentrations, the activation of the ChREBP activity occurs at two levels: translocation into the nucleus and transcriptional control [2]. ChREBP is a large transcription factor consisted of 865 amino acids with Mr = 96,500 kDa (Fig. 1A). It contains several functional domains including nuclear export signal site (NES), nuclear import signal sites (NLS), a DNA binding bHLH/Zip domain, and prolinerich regions implicated in protein–protein interactions including multiple phosphorylation sites [1,3]. Previous studies [4] have shown that the N-terminal region of ChREBP (residues 1–251) interacts directly with a dimeric form of the 14-3-3 protein, and Abbreviations: LPK, liver pyruvate kinase; ChREBP, carbohydrate response element binding protein; NES, nuclear export signal; NLS, nuclear localization signal; ChoRE, carbohydrate response element; CRM1, chromosome region maintenance 1; WT, wild-type. * Corresponding author. Address: Biochemistry Department, University of Texas Southwestern Medical Center, Dallas, TX 75390-9038, USA. Fax: +1 214 648 8856. E-mail address: [email protected] (K. Uyeda). 0006-291X/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.bbrc.2009.11.115
is responsible for sub-cellular cellular localization, while the C-terminal region of ChREBP forms a complex with a Max-like protein X (Mlx) to bind to various DNA sites, thereby activating the transcriptional activity [3,5]. Previous studies have reported a nuclear export signal site (NES), which is located at the amino acid residues (86–95) of ChREBP [6]. In this communication we report an identification of a new leucine-rich NES site located at the N-terminus of ChREBP (residues 5–15), and the effects of site-directed mutations of the site on the nuclear localization and transcription activities in hepatocytes and in vitro binding of ChREBP-14-3-3 complex to CRM1 (exportin). The results demonstrate that both NES sites are essential for the translocation of ChREBP from nucleus into cytoplasm. We propose to call the new site ‘‘NES 1” and change the previously identified ‘‘NES” site to ‘‘NES 2”. Materials and methods Chemicals. All the reagents were purchased from Sigma unless otherwise stated. The 14-3-3 antibodies (K-19) were purchased from Invitrogen (Carlesbad, CA). CRM1 IgG and Lipofectamine were products of Santa Cruz (Santas Cruz, CA). The expression vector for GST-tagged human CRM1 (hCRM1) was a gift of Dr. Y. M. Chook (Pharmacology, UT Southwestern Med. Cent., Dallas, TX). His-tagged 14-3-3b, GST-tagged human CRM1 (hCRM1) and GST-tagged rat CRM1 (rCRM1) were expressed in Escherichia coli and purified as described previously [4].
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Fig. 1. (A) A schematic representation of the functional domains and phosphorylation sites for cAMP-dependent protein kinase of ChREBP. The locations of the functional domains are shown: Nuclear export signal sites, NES1, NES2; nuclear localization signal site, NLS; proline-rich region, bHLH/ZIP, and ZIP-like domains. The locations of three phosphorylation sites of PKA are indicated as P1, P2, and P3. (B) Alignment of N-terminal region of ChREBP from rat, mouse and human. The NES and NLS sequences are boxed. Two NES regions contain the amino acid sequences fit consensus F–x2–3–F–x2–3–F–x–F (F, hydrophobic amino acids; x is any amino acid). Point mutations used in this study are also indicated above the original amino acids. P1 is PKA-phosphorylation site (Ser196), and an alpha helix (a2 Helix) is 14-3-3 binding site.
15 min at 22 °C and finally washed with PBS containing 50 mM glycine. The GFP-derived fluorescence was observed and analyzed using a confocal laser microscope. Typically three sets of about 100 fluorescent cells were counted and scored for sub-cellular localization [2]. Electrophoresis mobility-shift assay (EMSA). Gel mobility-shift assays were performed as previously described [7]. ChREBP antiserum (Novus) was added to the reaction mixtures for super-shift assays. Double-stranded oligonucleotides were prepared by mixing equal amounts of the complementary single-stranded DNAs in 50 mM NaCl, heating to 70 °C for 15 min, and cooling to 22 °C. The annealed oligonucleotides were labeled with 32P in the presence of [c32P]ATP (Amersham Pharmacia) and T4 polynucleotide kinase (New England Biolabs). Binding reactions were carried out in a reaction mixture containing 20 mM Hepes (pH 7.9), 50 mM KCl, 5 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF, 10% glycerol, 1.25 lg poly(dI-dC) (Amersham Pharmacia), and 10–25 lg nuclear extract. The reaction mixture was incubated at 22 °C for 30 min, and DNA–protein complexes were separated by electrophoresis in a non-denaturing 4.5% PAGE gel. Protein–protein interactions of ChREBP with 14-3-3 and CRM1 (Exportin). In vitro binding assays of interaction between ChREBP or mNES-ChREBPs, 14-3-3b and CRM1 were carried out as follows. FLAG-tagged ChREBP expressed in HEK293T and purified from cell lysate by incubating with beads bearing anti-FLAG antibodies as described before [4]. In the binding buffer, the FLAG-ChREBPbound beads were incubated with either homogeneous hCRM1 or rCRM1 (3 lg) and 14-3-3b (2 lg) in the reaction mixture (0.5 ml) containing 20 mM HEPES (pH 7.3), 110 mM KOAc, 2 mM MgCl2, 5 mM NaOAc, 0.5 mM EGTA, and 0.01% Nonidet P-40 for 1.5 h at 4 °C with gentle rocking. After washing the beads (3 times) with reaction buffer without serum albumin, proteins were eluted with SDS–PAGE sample buffer. Samples were then subjected to PAGE and immunoblot detection for CRM1, 14-3-3, and ChREBP. Results
Construction of plasmids. Construction of expression vectors for ChREBP, GFP-tagged ChREBP, and FLAG-tagged ChREBP were described previously [4]. Oligonucleotides used for site-directed mutagenesis were; mNES1, 50 -ATGGCGCGCGCGGCGGCGGATCTA TCCGTGAACGCGCAGGTCCCCGG-30 and 50 -TccggggacctgcgcgttcaCG GATAGATCCGCCGCCGCGCGCGCGCCAT-30 . Immunoblot analysis. Protein samples were separated on either on an 8.0 % or a 10.0% SDS–PAGE gel and blotted onto Trans-Blot nitrocellulose membrane (Bio-Rad). The trans-blots were briefly rinsed with Tris-buffered saline (TBS) buffer containing 0.1% Tween-20 (TBS-T) then incubated for 1 h at 22 °C in TBS-T containing 5% non-fat milk (Difco). The blocked membrane was incubated with primary antibodies for 1 h at 22 °C. Secondary antibody, horseradish peroxidase-conjugated anti-rabbit IgG (ZyMed) were used as positive signals, which were visualized by ECL western blotting detection system (Amersham). Primary hepatocyte culture, transfection, and luciferase reporter assay. Isolation of hepatocytes by collagenase treatment from rat liver, transfection with Luciferase reporter and sub-cellular localization were performed as described before [4]. Sub-cellular localization of ChREBP. To analyze the sub-cellular localization of ChREBP, primary hepatocytes were plated onto 35 mm glass-bottomed dish (MatTek) coated with type I collagen at a density of 5.0 105 per dish. Onto the hepatocyte-containing plates either wild-type or the mutated (3.2 lg) forms of GFP-ChREBP-expressing plasmids were transfected by Lipofectamine 2000 (see above). After 16 h transfection 27.5 mM glucose was added for the high glucose condition, and after 24 h the cells were washed once with PBS, fixed with PBS buffered with 4% formaldehyde for
Mutations at either NES1 or NES2 sites affect ChREBP binding to CRM1 A computer search revealed a new leucine-rich nuclear export signal (NES) site, termed ‘‘NES1”, located at the N-terminus of mNES1 WT
mNES1/2
mNES2
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14-3-3
FLAG-ChREBP
Fig. 2. CRM1 and endogenous 14-3-3 bound to FLAG-ChREBP and mutants of NES (NES1, NES2 and NES1/2). Wild-type FLAG-ChREBP, mutants of NES1, NES2 and NES1/2 of FLAG-ChREBP were expressed in HEK293 cells and the expressed ChREBP were bound to anti-FLAG antibodies beads. The CRM1 and 14-3-3 bound to the beads were eluted and analyzed by PAGE and subjected to Western blotting as described under the Materials and methods section.
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ChREBP (5LADLSVNLQVP15), in addition to the previously reported ‘‘NES” site (85TLTRLFECLSLA95) [3]. In order to determine the role of NES1, we have prepared a mutant of the NES1 site (mNES1) by substituting two leucine residues with alanines (L5A and L12A). For comparison, we have employed the previously described mNES2 [4]. The wild-type-ChREBP (WT) and the mutants of both NES sites of ChREBP were expressed in HEK293 cells, following expression cell extracts were incubated with homogeneous rat CRM1 (hCRM1) (Fig. 2). Wild-type (WT)-ChREBP bound hCRM1 as well as the endogenous 14-3-3 proteins were tested for binding under these conditions (Fig. 2). Both mNES1- and mNES2-ChREBP also bound to CRM1 and to the endogenous 14-3-3, but by much less amounts compared to that with the WT-ChREBP. However, mutation of both NES sites (NES1 and NES2) of ChREBP completely abolished the CRM1 binding to ChREBP. The same results were obtained with the rat CRM1 protein. These results indicate that both NES1 and NES2 sites are required for maximum binding of CRM1. It is also worth noting that the amounts of the 14-3-3 proteins bound to WT-ChREBP and the individual NES mutants (either NES1 or
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NES2) of ChREBP were similar to those of CRM1 binding alone, suggesting that CRM1 may interact with ChREBP-14-3-3 heterodimer. Furthermore, these results suggest that the HEK293 cells contain sufficient endogenous 14-3-3 in the cells to form a stable ternary complex. In the export of proteins usually Ran GTPase and GTP are required [8], but the addition of an active form of purified Ran Q69L-GTPase [8] and GTP to the binding reaction mixture had no effect. The effects of mNES1 and mNES2 on the nuclear localization and transcriptional activities of ChREBP In order to further elucidate the roles of NES1 for export of ChREBP from the nucleus to the cytoplasm, we have determined the sub-cellular localization of GFP-ChREBP, and all three NES mutants in primary hepatocytes using confocal microscopy. GFP-tagged WT-ChREBP localized exclusively in the cytosol under low glucose conditions, but under high glucose ChREBP migrated to the nucleus (Fig. 3A and B). However, GFP-mNES1ChREBP was localized to the nucleus, either under low or high
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40
20
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mNES1
mNES2
mNES1/2
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mNES2
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Fig. 3. (A) Sub-cellular localization of full-length ChREBP and its mutants the NES or NLS in primary rat hepatocytes under low glucose and high glucose condition. Primary cultured rat hepatocytes were co-transfected with GFP-tagged ChREBPs. The cells were cultured in D-MEM containing 5.5 mM glucose, and then incubated for additional 8 h with 5.5 mM (open bars) or 27.5 mM (filled bars) glucose-containing medium. Cells were fixed with 4% formaldehyde and localization of fluorescence was counted under confocal microscopy. The values presented are the mean and SD of three sets of about 100 fluorescent cells. mNES1, mNES2, mNES1/2 and mNLS mutations were introduced within NES1, NES2, both of NES1 and NES2, and NLS, respectively. (B) Representative picture of sub-cellular localization of wild-type and the NES mutants of ChREBP used in (A) under low glucose condition. (C) The effect of mutation of the NES sites on glucose-responsive transcriptional activity. Primary cultured rat hepatocytes were co-transfected with the ChREBP expression vectors as described in Materials and methods and the cells were incubated for 12 h in low and high glucose as above. Luciferase reporter activities were measured and are shown as the firefly luciferase activity [1]. The values presented are the mean and SD of replicate (three to four) in cultures from a single determinations represent more than three individual experiments. Total amount of DNA for expression vector was compensated with empty vector, pcDNA3.1.
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glucose conditions. A similar nuclear localization was observed with mNES2, confirming our previous results [4]. GFP-mNES1/ 2-ChREBP also remained in the nucleus. However, unlike the NES mutants, a mutant of the nuclear localization signal site (NLS) mNLS-ChREBP was unable to localize ChREBP to the nucleus, as expected (Fig. 3A). These results demonstrated the importance of both NES1 and NES2 sites for export of ChREBP out of nucleus into the cytosol, because unlike WT-ChREBP these NES site mutants were unable to exit the nucleus even under low glucose conditions. The transcriptional activity assay of ChREBP in which the ChREBP-activated LPK promoter drove the expression of the luciferase reporter [1,4] revealed that WT-ChREBP was activated 3-fold greater under high glucose conditions as compared to low glucose conditions (Fig. 3C). However, mNES1-ChREBP showed approximately 36% lower activity in high glucose when compared to that of the WT-ChREBP and NES2 and NES1/2 both failed to show any transcriptional activity. In contrast to the nuclear localization activities, the differences in the transcriptional activities of the mNES1-ChREBP under high and low glucose (‘‘glucose sensitivity”) was retained, but the glucose activation was decreased approximately 30% compared to the WT-ChREBP. The transcriptional activities of the other mutants of the mNES2-ChREBP and that mNES1/2-ChREBP were completely lost. These results suggest that either the sub-cellular distribution assay is less sensitive to glucose concentration than the transcriptional activity or that some of the NES1 mutant remained in the nucleus, thereby retaining the transcriptional activity. Although the NES1/2-ChREBP localized in the nucleus, all transcriptional activity was lost. Further examination of this unexpected loss of the transcription activity of the ChREBP double mutant in spite of nuclear localization, revealed that the mNES1/2-ChREBP possessed DNA binding activity as demonstrated by the super-shift assay. The mutation of both NES sites showed additive effects on the nuclear localization of GFP-tagged ChREBP. NES1 and perhaps NES2 sites may play a role in glucose sensing since they both inactivated nuclear export and decreased glucose sensitivity. The export of CRM1 usually binds its cargo protein in the presence of Ran GTPase and GTP in the export [9]. However, Ran GTPase is not required for CRM1 binding of ChREBP as is in CRM1-SNUPN complex [10]. Discussion In response to increased circulating glucose, ChREBP is shifted from the cytoplasm to the nucleus, thereby down-regulating LPK expression and controlling lipogenic genes, which regulate fat storage. Under low glucose conditions such as starvation the transcriptional activity of ChREBP is terminated probably by phosphorylation mediated by protein kinases such as PKA, followed by interaction with 14-3-3, finally the ChREBP-14-3-3 complex binds to CRM1 to localize the ternary complex in the cytosol. Here we present a new export signal site of ChREBP termed ‘‘NES1” in addition to the previously reported ‘‘NES” site [6], now renamed ‘‘NES2”. The NES1 site (residues 5LADLSVNLQVP15) is located in the N-terminus of ChREBP. The evidence in support of this suggestion is based on the following results: the substitution of two Leu residues with Ala resulted in (a) the decreased CRM1
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binding in the in vitro ‘‘pull-down” assays, (b) a loss of the nuclear localization of GFP-tagged ChREBP mutants, and (c) a loss of transcriptional activity as determined using a reporter activity assay. It appears that both NES1 and NES2 sites are required for the maximum interaction with CRM1, and the maximum nuclear export and the transcriptional activities of ChREBP. Mutations in either the NES1 or the NES2 site alone affected ChREBP binding only partially. The amino acid sequnces of the nuclear export signal site (NES1) of ChREBP as well as NES2 do not seem to fit the consensus sequence of Ø–X(2–3)–Ø–X(2–3)–Ø–X–Ø (in which Ø is Lys, Val, Ile, Phe or Met, and X is any amino acid [10,11]. Moreover, recently the crystal structure of CRM1 bound to snurportin 1 (SNUPN) has been reported [10,12]. The amino acid sequence of ChREBP NES1 includes Met-1, Leu-5, Leu-8, Leu-12, and Val-14, which is nearly identical to that of SNUPN. According to the 3-D structure the N-terminal NES peptide of SNUPN is an a-helical-extended structure that binds to a highly conserved hydrophobic groove of CRM1 outer helices. Due to the sequence similarity, it is likely that the NES1 site in ChREBP possesses a combined helix-extended structure, which may also bind to the same NES-binding groove of CRM1. The second NES site of SNUPN is mainly basic and binds to an acidic surface of CRM1, which is situated adjacent to the first NES site. In contrast to the SNUPN, the NES2 epitope of ChREBP has one basic and one acidic residue, consequently it may not bind adjacent to NES1. However, the mutational effects on NES1 and NES2, in ChREBP, appear to be additive on all three biological activity assays performed here. This may favor both NES epitopes binding adjacent to each other, similar to the SNUPN example. Acknowledgments This work was supported by grants from the Department of Veterans Affairs and by National Institute of Health Grant DK063948. We thank Dr. Yuh M. Chook (Univ. Texas Southwestern Med. Cent., Dallas, TX) for the hCRM1 DNA and her valuable discussion. References [1] H. Yamashita, M. Takenoshita, M. Sakurai, R.K. Bruick, W.J. Henzel, W. Shillinglaw, et al., Proc. Natl. Acad. Sci. USA 98 (2001) 9116–9121. [2] T. Kawaguchi, M. Takenoshita, T. Kabashima, K. Uyeda, Proc. Natl. Acad. Sci. USA 98 (2001) 13710–13715. [3] S. Cairo, G. Merla, F. Urbinati, A. Ballabio, A. Reymond, Hum. Mol. Genet. 10 (2001) 617–627. [4] H. Sakiyama, R.M. Wynn, W.R. Lee, M. Fukasawa, H. Mizuguchi, K.H. Gardner, et al., J. Biol. Chem. 283 (2008) 24899–24908. [5] A.K. Stoeckman, L. Ma, H.C. Towle, J. Biol. Chem. 279 (2004) 15662–15669. [6] G. Merla, C. Howald, S.E. Antonarakis, A. Reymond, Hum. Mol. Genet. 13 (2004) 1505–1514. [7] S. Ishii, K. Iizuka, B.C. Miller, K. Uyeda, Proc. Natl. Acad. Sci. USA 101 (2004) 15597–15602. [8] D. Gorlich, U. Kutay, Annu. Rev. Cell. Dev. Biol. 15 (1999) 607–660. [9] D. Gorlich, N. Pante, U. Kutay, U. Aebi, F.R. Bischoff, EMBO J. 15 (1996) 5584– 5594. [10] X. Dong, A. Biswas, K.E. Suel, L.K. Jackson, R. Martinez, H. Gu, et al., Nature 458 (2009) 1136–1141. [11] U. Kutay, S. Guttinger, Trends Cell Biol. 15 (2005) 121–124. [12] T. Monecke, T. Guttler, P. Neumann, A. Dickmanns, D. Gorlich, R. Ficner, Science 324 (2009) 1087–1091.