Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of Threonines 222/226
© Liu et al; licensee BioMed Central Ltd. 2006
Received: 09 November 2005
Accepted: 06 April 2006
Published: 06 April 2006
Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoeno lpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) gene expression. CAAT/enhancer binding protein alpha (C/EBPα) regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226) by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBPα is a link between GSK3 and these gene promoters.
C/EBPα represses the IGFBP1 thymine-rich insulin response element (TIRE), but mutation of T222 or T226 of C/EBPα to non-phosphorylatable alanines has no effect on C/EBPα activity in liver cells (towards the TIRE or a consensus C/EBP binding sequence). Phosphorylation of T222/T226 is decreased by GSK3 inhibition, suggesting GSK3 does phosphorylate T222/226 in intact cells. However, phosphorylation was not altered by treatment of liver cells with insulin. Meanwhile C/EBPα activity in 3T3 L1 preadipocytes was enhanced by mutation of T222/T226 and/or S230 to alanine residues. Finally, we demonstrate that C/EBPα is a very poor substrate for GSK3 in vitro and in cells.
The work demonstrates an important role for this domain in the regulation of C/EBPα activity in adipocytes but not hepatocytes, however GSK3 phosphorylation of these residues does not mediate regulation of this C/EBP activity. In short, we find no evidence that C/EBPα activity is regulated by direct phosphorylation by GSK3.
Glycogen synthase kinase-3 (GSK3), an insulin-inhibited protein kinase, has been linked to the control of many cellular processes . It was originally identified as the protein kinase that phosphorylated and inactivated glycogen synthase in rabbit muscle . Two highly related forms of GSK3 (GSK3α and GSK3β) are expressed from distinct genes [3, 4]. They share greater than 95% identity in their kinase domains and appear to be ubiquitously expressed in mammals [3, 4]. In resting cells GSK3 activity is high and inhibition of the kinase is achieved by at least two mechanisms, firstly through phosphorylation of an N-terminal serine residue (Ser-21 in GSK3α, Ser-9 in GSK3β) [5, 6], and secondly through protein protein interaction . Insulin promotes the phosphorylation of Ser-9/21 of GSK3 by activation of protein kinase B (PKB, also known as c-AKT) , while canonical wnt signalling inhibits GSK3 independently of this N-terminal phosphorylation .
In liver, inhibition of GSK3 reduces expression of the Phosphoenolpyruvate Carboxykinase (PEPCK) and Glucose-6 Phosphatase (G6Pase) genes, two rate limiting enzymes in hepatic glucose production , as well as the IGF binding protein-1 (IGFBP1) gene . These gene promoters share a related DNA sequence termed the thymine-rich insulin response element (TIRE) that plays an important role in their response to insulin [12–14]. Expression of these genes as well as GSK3 activity is abnormally high in insulin resistant states such as Type 2 diabetes mellitus (T2DM) [15–19]. Meanwhile overexpression of GSK3 antagonises insulin regulation of IGFBP1 . Therefore inhibition of GSK3 is proposed as a potential target for the treatment of T2DM [1, 20, 21]. Indeed selective GSK3 inhibitors significantly reduce hepatic glucose output and blood glucose levels in several animal models of obesity and diabetes [22–25]. However, GSK3 is reported to have over 30 substrates, and mutation of one of these, APC, is linked to the development of colonic cancer [20, 21, 26]. In addition, genetic deletion of one of the isoforms (GSK3β) leads to liver cell death, possibly due to increased sensitivity to TNFα . Hence great care and consideration is being given to the development of GSK3 inhibitors for use in humans. With this in mind we have initiated a search for molecules that link GSK3 to the gluconeogenic gene promoters and in particular the TIRE, since inhibition of GSK3 is sufficient to repress the isolated DNA element . Identification of the GSK3 substrate(s) that regulates IGFBP1, G6Pase and PEPCK gene expression, may permit manipulation of this specific interaction without affecting other GSK3 targets, thereby reducing potential side effects. GSK3 is an unusual kinase in that most substrates must be phosphorylated (primed) on a Ser/Thr four or five residues C-terminal to the GSK3 target residue (see  for review). Priming of the substrate is performed by a distinct protein kinase, therefore inhibition of the priming kinase indirectly inhibits GSK3 regulation of the substrate, but only those substrates primed by that particular priming kinase. Priming kinases are potential targets for more specific interference with GSK3 function.
The basic leucine zipper transcription factor, CAAT-enhancer binding protein alpha (C/EBPα) is a potential link between GSK3 and the gluconeogenic genes based on the following information in the literature; 1) C/EBPα is phosphorylated on two residues (T222 and T226, numbers refer to rodent sequence) by GSK3, although the functional effect of phosphorylation is not yet clear , 2) C/EBPα is a key molecule in the regulation of the PEPCK and G6Pase gene promoters by cAMP [29–33] and T3 , 3) C/EBPα is regulated by wnt  and insulin [36, 37] in brown adipocytes, and the insulin regulation involves PKB (a regulator of GSK3) , 4) PEPCK and G6Pase expression, as well as glucose and glycogen metabolism is abnormal in animals lacking C/EBPα [38–40] and 5) binding of a TIRE-interacting protein is blocked using an oligonucleotide representing a known C/EBPα binding sequence .
Therefore, we attempted to determine whether C/EBPα could modulate the activity of the isolated TIRE, or the gene promoters of interest, in a GSK3 regulated manner. In addition, although T222/T226 of C/EBPα were identified as targets for GSK3 it has not previously been established whether S230 was a target for a 'priming' kinase, so we have also investigated whether mutation of S230 influences C/EBPα regulation.
Results and discussion
C/EBPα regulates the TIRE independently of T222/226 phosphorylation
GSK3 phosphorylates C/EBPα in intact cells
C/EBPα is a relatively poor substrate for GSK3 in vitro
GSK3 phosphorylation of T222/226 in cells does not require S230 phosphorylation
Primers and oligonucleotides used in C/EBP mutagenesis. Primers and oligonucleotides
Forward: 5'-GGA TCC GCC ACC ATG GAC TAG AAG GAC GAC GAT GAC AAG GAG TCG GCC GAC TTC TAC GAG-3'
Reverse: 5'- GAA TTC TCA CGC GCA GTT GCC CAT GGC CTT GAC-3'
S230A Flag-C/EBPα mutant
Sense: 5'-CGC CCG TGC CCG CCC CTC ATC CCG-3'
Antisense: 5'-CGG GAT GAG GGG CGG GCA CGG GCG-3'
Sense:5'-GGC CAC CCT GCG CCG CCG CCG GCG CCC GTG CCC AGC CCT CAT CCC-3'
Antisense: 5'-GGG ATG AGG GCT GGG CAC GGG CGC CGG CGG CGG CGC AGG GTG GCC-3'
Forward: 5'-GGA TCC GCC ACC ATG AGC GGC GGC GGG CCT T-3'
Reverse: 5'-TCT AGA GGA AGA GTT AGT GAG GGT AGG TGT GGCA-3'
Sense:5'-CGG GGT ACC CCG ATT TTT GCG CAA TTT TAT TGC GCA ATC AAT ATT GGA AGA TCT TC- 3'
Antisense: 5'-GAA GAT CTT CCA ATA TTG ATT GCG CAA TAA AAT TGC GCA AAA ATC GGG GTA CCC CG-3'
Comparison of mouse, rat and human C/EBPα sequences surrounding potential GSK3 target residues. Numbers refer to mouse (upper) and human (lower) potential phosphorylated residues. C/EBPα sequences.
222 226 230
PGHPT PPPT PVPS PHAA
PGHPT PPPT PVPS PHPA
PGHPT PPPT PVPS PHAA
226 230 234
C/EBPα induced a consensus binding element while repressing the TIRE in H4IIE cells
C/EBPα overexpression does not regulate endogenous TIRE-containing genes
C/EBPα regulation of the CBE in preadipocytes is greater when T222/226 are mutated
In summary, C/EBPα is not the link between GSK3 and the hepatic TIRE-containing gene promoters. However, C/EBPα can regulate the TIRE, at least when overexpressed, and the residues T222/226 and S230 all influence C/EBPα function in preadipocytes. Interestingly we demonstrate opposite effects of C/EBPα overexpression on TIRE and CBE activity, and a tissue specific role for the T222/226 motif in this activity. Our data also provides further evidence of the differences in transcription factor activity when measured using isolated promoter elements as opposed to intact gene promoters. During the course of this study a regulation of C/EBPβ by GSK3 in adipocytes was reported . Therefore it will be of interest to confirm whether this C/EBP isoform is the link between GSK3 inhibition and TIRE regulation in hepatocytes.
Radioisotopes [γ-32P]ATP (Amersham Biosciences, Inc., Little Chalfont, Buckinghamshire, UK) and [α-32P]UTP (ICN, Thame, Oxfordshire, UK) were purchased from the indicated sources. Insulin was obtained from Novo Nordisk (Crawley, West Sussex, UK), dexamethasone and IBMX (Sigma, Poole, Dorset, UK), RNAse Protection Assay Kit II was from AMS Biotech/Ambion (Austin, TX, USA). CHIR99021 was synthesised by Dr Rudolpho Marquez . Antibodies to the following epitopes were purchased from the companies in parenthesis; C/EBPα (Santa Cruz Biotechnologies, UK), phospho-C/EBPα (T222/226) and phospho-GSK3α/β (S21/9) (Cell Signaling Technologies, Hertfordshire, UK), Flag (Sigma, Poole, Dorset, UK), while the Glycogen Synthase (total and site 3 phospho antibody), and p90 RSK phospho antibody were made in the Division of Signal Transduction Therapy, University of Dundee.
The rat hepatoma cell line H4IIE was cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 1000 mg/L glucose, 5% (v/v) foetal calf serum (GIBCO, Carlsbad, CA). The AD293 cell line was maintained in DMEM containing 4500 mg/L glucose, 10% (v/v) foetal calf serum, while 3T3-L1 preadipocytes were grown in DMEM containing 4500 mg/L glucose and 10% (v/v) newborn calf serum (GIBCO).
Construction of plasmid DNA, luciferase reporter, and mutagenesis
The full length C/EBPα cDNA was amplified by PCR from a rat L6 cDNA library and BamHI/Kosac/Flag and EcoRl sequences introduced at the 5' and 3' end respectively as indicated in Table 1. The PCR product was subcloned into TOPO one shot (Invitrogen), verified by DNA sequencing, and subcloned into pcDNA6, GST-pGEX-6, and pshuttle-CMV vectors for the purpose of expression in mammalian cells, production of recombinant GST-tagged protein in Escherichia coli BL21 cells, and construction of recombinant adenovirus (AdEasy XL Adenoviral Vector System, Stratagene), respectively. Flag-C/EBPα point mutants, S230A, T222A/T226A (AA), and T222A/T226A/S230A (AAA) were generated using the QuickChange mutagenesis kit (Stratagene) with the oligo sequences shown in Table 1. The AAA mutant was generated by introducing the S230A point mutation into the AA mutant. A luciferase reporter construct containing a C/EBP consensus binding sequence (CBE-Luc) 5' of a thymidine kinase promoter was constructed using the oligos shown in Table 1. Both sense and antisense oligos were annealed prior to restriction digest with Kpnl and Bgl II. The product was subcloned into the BP1 luciferase reporter linearised with Kpnl and BamHI to remove the TIRE (the luciferase construct was a gift from Dr Robert Hall and Professor Daryl K. Granner (Vanderbilt University, TN, USA)).
AD293 cells were transiently transfected with 10 μg of pcDNA6, Flag-C/EBPα, AA, AAA, or S230A mutants by the calcium phosphate method. DNA precipitant was added to cells for 4 h, prior to addition of inhibitors and hormones at the concentrations and for the times indicated in figure legends.
H4IIE cells were transfected as described previously . Briefly, 10 μg of BP1-TIRE-Luc, the insulin insensitive point mutant of the TIRE (DM5-Luc), C/EBP-Luc, or pGL3-Luc (control), along with 1 μg of pcDNA6, wild type or mutant Flag-C/EBPα constructs were precipitated and incubated with H4IIE cells in suspension. Cells were plated onto 10 cm dishes, allowed to attach for 4 h, prior to DMSO shock and incubation with hormones/inhibitors for 20 h as indicated in figure legends. Cells were lysed in 200 μl lysis buffer (Promega, UK), the cell debris removed by centrifugation at 13,000 × g for 10 min at 4°C, and the supernatant stored at -70°C. Luciferase assays were performed using the firefly luciferase assay system (Promega, UK), according to manufacturer's instructions, with luciferase activity being corrected for the protein concentration in the cell lysate.
3T3-L1 preadipocytes were transfected using Fugene 6 Transfection Reagent (Roche) following manufacture's protocol. C/EBP-Luc (2 μg) along with 200 ng of pcDNA6, wild type or mutant Flag-C/EBPα constructs was complexed with Fugene 6 transfection reagent prediluted in Serum Free DMEM at the ratio of 1:3 (μg DNA : μl Fugene 6) for 20 min at RT before addition to cells. After 4 h at 37°C an equal amount of DMEM containing 20% foetal calf serum was added. 48 h post-transfection, cells were washed with serum free DMEM and incubated for a further 24 h in serum free media with or without hormones or inhibitors as described in figure legends. Cells were lysed in 100 μl lysis buffer and luciferase activity determined as above.
We estimate that the level of over expression of C/EBPα is roughly 4 fold in the adipocytes, around 6-fold in the liver cells by transient transfection and nearly 10 fold in adenovirally infected H4IIE experiments
In vitro phosphorylation of C/EBPα
Purified recombinant C/EBPα (6 pmol) was incubated at 30°C with His6-GSK3β (2 U/ml) or p42 MAPK (10 U/ml), 10 mM magnesium acetate, 0.1 mM [γ-32P]ATP (450,000 cpm/nmol) in buffer containing 50 mM Tris-HCl, pH 7.5, 0.03% (v/v) Brij 35, and 0.1% (v/v) 2-mercaptoethanol, for times indicated in the figure legends. Reactions were stopped by the addition of LDS containing sample buffer (Novex), heated at 70°C for 10 min and subjected to SDS-PAGE. The gel was dried on the 3 M Filterpaper, and radioactivity incorporated into recombinant C/EBPα visualized by autoradiography and quantified by phosphorimager (Fuji). A standard curve of radiolabelled ATP was used to assess nmoles of phosphate incorporated into the C/EBPα substrate. p42 MAP kinase and GSK3 activities were calculated by in vitro phosphorylation of myelin basic protein and P-GS peptide, respectively.
Preparation of cell extract for SDS PAGE and Immunoblotting
Cells were washed with ice cold PBS twice and scraped into ice-cold lysis buffer (25 mM Tris/HCl, pH 7.4, 50 mM NaF, 100 mM NaCl, 1 mM sodium vanadate, 5 mM EGTA, 1 mM EDTA, 1% (v/v) Triton X-100, 10 mM sodium pyrophosphate, 1 mM benzamidine, 0.1 mM PMSF, 0.27 M sucrose, 2 μM microcystin and 0.1% (v/v) 2-mercaptoethanol). Cell debris was removed by centrifugation at 13,000 × g for 10 min at 4°C. Preparation of nuclear protein extracts was performed using a Nuclear Extraction Kit (Panomics), as per the manufacture's instructions. The protein concentration was determined by Bradford assay (Sigma), using BSA as standard as per the manufacture's instructions. Protein from cell lines (10–20 μg from H4IIE, 50 μg from AD293 or 3T3 preadipocytes) was separated on Novex SDS 4–12% polyacrylamide gels. Following transfer to nitrocellulose, blots were blocked with 5% (w/v) non-fat milk in TBST (Tris-buffered saline containing 0.1% (v/v) Tween 20) for 1 h, and incubated with primary antibodies at 4°C overnight prior to incubation for 1 h at RT with the secondary antibody and development using ECL kit (Amersham Biosciences, Inc.).
Adenoviral Infection, RNA extraction and RNAse protection assay
For RNA studies, H4IIE cells were infected with recombinant adenovirus expressing Flag-C/EBPα (Ad-C/EBPα) or β-Galactosidase (Ad-β-gal) for 4 h at MOI = 50. 24 h later, infected cells were split into 10 cm dishes and serum starved overnight prior to treatment with hormone/inhibitor for 3 h at the concentrations indicated in the figure legends. Total cellular RNA was isolated using TRI Reagent (Sigma) following the manufacturer's instructions. An RNase Protection Assay (RPA) was performed to determine the relative amounts of IGFBP-1, G6Pase, and cyclophilin mRNA as described previously . Band intensity was quantified on a phosphorimager (Fuji), data calculated as a ratio of IGFBP-1 to cyclophilin mRNA. For phosphorylation studies, H4IIE cells were infected with recombinant adenovirus expressing Flag-C/EBPα (Ad-C/EBPα) at MOI = 100. 16 h later, infected cells were serum starved for 3 h prior to treatment with hormone/inhibitor for the times and at the concentrations indicated in the figure legends.
The comparison of one variable between two groups was determined by unpaired Student's t-test with the aid of PRISM 3.0 (Graphpad Software, USA) and/or Microsoft Excel. The values were expressed as mean ± S.E.M.
IGF binding protein-1
RNAse Protection Assay
thymine rich insulin response element
CAAT enhancer binding protein
C/EBP binding element
CL is a recipient of a BBSRC CASE studentship while CS is a recipient of the Diabetes UK Senior Fellowship (BDA:RD02/0002473). This work was primarily supported by Diabetes UK grant (BDA:RD03/0002583).
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