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Shinsaku Aramata,
Kunio Yasuda, and
Kohsuke Kataoka*
Laboratory of Molecular and Developmental Biology, Graduate School of Biological Science, Nara Institute of Science and Technology, Ikoma, Japan
Received 23 August 2006/ Returned for modification 22 September 2006/ Accepted 23 July 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pdx1 is a homeodomain transcription factor that binds AT-rich nucleotide sequence motifs (termed A1, A3, and GG2 elements) in the insulin promoter (21, 27, 34, 38). Beta2 is a basic helix-loop-helix factor that binds as a heterodimer with the ubiquitously expressed E47 protein to the E1 element (31). Gene disruption experiments with mice have revealed that both Pdx1 and Beta2 play critical roles in insulin gene expression as well as in islet development and function (1, 30). Furthermore, mutations in pdx1 and beta2 are found in patients with maturity onset diabetes of the young type 4 and type 6, respectively, and in some populations of patients with type 2 diabetes (12, 23, 26, 47).
Previous studies have shown that Pdx1 and Beta2 are also involved in glucose-regulated insulin promoter activation and that the DNA-binding activities of these factors to their respective cis elements is induced by glucose (8, 25, 27). It has also been demonstrated that a rise in glucose concentration induces nuclear translocation and increases the transactivation capacity of Pdx1 and Beta2 (6, 24, 36, 37, 40). However, the precise molecular mechanisms by which these proteins are activated are not fully understood.
Another cis-regulatory element, termed RIPE3b/C1, has been shown to be important not only for ß-cell-restricted expression of the insulin gene but also for its up-regulation by glucose (42-44). Mutation of the C1 element abolished the glucose responsiveness of the insulin promoter in reporter assays. In addition, C1-binding activity in ß-cell nuclear extracts, detected by gel mobility shift assay, increased in response to glucose. A ß-cell-specific transcription factor, MafA, was subsequently identified as the C1-binding factor (15, 17, 28, 35).
MafA is a member of the Maf family of basic leucine zipper (bZip) transcription factors, which also includes MafB, c-Maf, and Nrl. MafA, together with Pdx1 and Beta2, synergistically activates the insulin promoter (2, 5, 55), and coexpression of these three factors in non-ß cells induces endogenous insulin expression (16). Recently, it was also shown that ablation of mafA in mice leads to a deficiency in glucose-stimulated insulin secretion from ß cells, progressive ß-cell degeneration, and diabetes (54).
Despite this progress in characterizing MafA function, the mechanism of MafA regulation by glucose remains unknown. In this study, we examined the phosphorylation status of MafA in ß cells and examined its relationship to the biological activity of MafA and to the regulation of MafA by glucose. We demonstrated that MafA protein stability is regulated by glucose. In ß cells, MafA is constitutively phosphorylated at multiple sites by glycogen synthase kinase 3 (GSK3) and is rapidly degraded under low-glucose conditions but not under high-glucose conditions.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bsu36I was constructed by double digestion of pHygEF2/m-mafA (17) with ClaI (located in the multicloning site) and Bsu36I (located in the mafA open reading frame), followed by blunting and self-ligation. To construct the IRES-EGFP cassette plasmid, a NotI-NcoI fragment from pUC/NotI/IRES (20), containing the internal ribosome binding site (IRES) of encephalomyocarditis virus, and an NcoI-NotI fragment from pEGFP-1 (Clontech), containing the entire open reading frame of enhanced green fluorescent protein (EGFP), were ligated and inserted into the NotI site of pBluescript II SK(–). The NotI fragment was then excised and inserted into the unique NotI site of pHygEF2 and pHygEF2/HA-m-mafA (the wild type [WT], the 4A mutant [S49A T53A T57A S61A], or the 5A mutant [S49A T53A T57A S61A S65A]) to generate pHygEF2/IRES-EGFP and pHygEF2/HA-m-mafA (WT, 4A, or 5A)-IRES-EGFP, respectively.
pHygEF2/HA-h-mafA, pHygEF2/FLAG-Pdx1, pHygEF2/FLAG-Beta2, h-ins-p-luc, and pEF-Rluc were described previously (17, 20). pG4x5/TATA/luc was a gift from Kazuhiko Igarashi (Tohoku University).
A DNA fragment containing the Gal4 DNA binding domain (DBD), Gal4-DBD, was amplified by PCR using the specific primers 5'-AGAGAATTCGTCATGAAGCTACTGTCTTCT-3' and 5'-AGAGGTACCCAAGCTTCGATACAGTCAACT-3' and pEF/Gal4-DBD (19) as the template. An amplified fragment was digested at the newly incorporated KpnI restriction site at its 3' end, ligated to the SacII site of m-mafA by blunt-end ligation, digested with EcoRI and BssHII, and inserted into the corresponding sites of pHygEF2. The resultant plasmid, pHygEF2/Gal4-mafA, encoded Gal4-DBD fused with amino acid residues 4 to 148 of MafA, with an additional His Arg construct at the carboxyl terminus.
U0126, MG132, and lactacystin were purchased from Calbiochem. Cycloheximide (CHX) (Sigma), lithium chloride (LiCl) (Nacalai tesque), and SB216763 (Tocris) were also obtained commercially.
Cells and transfection. MIN6 cells were a generous gift from Jun-ichi Miyazaki (Osaka University) (29). ßTC6 and NIT-1 cells were purchased from the American Tissue Culture Collection. The In1024 cell line was established from a BK virus-induced hamster insulinoma (48). MIN6, ßTC6, In1024, NIH 3T3, BHK21, and CHO cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% (for MIN6 and ßTC6 cells) or 10% (for the others) fetal calf serum. NIT-1 cells were grown in F12K medium supplemented with 10% fetal calf serum. The medium contained 25 mM glucose unless otherwise indicated. When glucose conditions were varied, the medium was changed to DMEM containing the indicated concentrations of glucose and supplemented with 10% fetal calf serum. Primary islets of Langerhans were isolated from 5-week-old Jcl:ICR mice (CREA Japan, Inc.) by a standard collagenase digestion method (9) and grown in RPMI containing 10% fetal calf serum.
For transfection, In1024, NIH 3T3, BHK21, or CHO cells grown in 24-well plates were transfected with a total of 0.8 µg of plasmid using 2 µl of Lipofectamine 2000 reagent (Invitrogen). For MIN6 cells, 3.2 µg of plasmid and 8 µl of Lipofectamine 2000 reagent were used.
Preparation of cell extracts and immunoblotting.
Nuclear extracts were prepared as described previously (41). Whole-cell extracts were prepared by direct addition of 4x sodium dodecyl sulfate (SDS) sample buffer (200 mM of Tris-HCl, pH 6.8, 8% SDS, 400 mM dithiothreitol, 0.2% bromophenol blue, 40% glycerol) to the cells or by cell lysis in radioimmunoprecipitation assay buffer (10 mM Tris-HCl, pH 7.5, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl) containing protease inhibitor cocktail (Nacalai tesque), followed by sonication for 20 s. For calf intestine alkaline phosphatase (CIAP) treatment, 20 U of CIAP (Takara) was added to aliquots of nuclear extracts (
1 to 2 µg) in 25 mM Tris-HCl, pH 7.9, 100 mM NaCl, 5 mM MgCl2, and 10 mM dithiothreitol and incubated at 30°C for 30 min. For metabolic labeling with 32P, a MIN6-derived stable cell line expressing HA-MafA-IRES-EGFP (clone no. 5) was grown in 12-well plates in DMEM containing 0.3 or 20 mM glucose for 12 h. Cells were washed with phosphate-free DMEM and then labeled by adding 0.5 mCi of [32P]orthophosphate (MP Biomedicals) for 6.5 h. For pulse and chase experiments, MIN6/HA-MafA-IRES-EGFP cells (clone no. 5) were grown in DMEM containing 0.3 or 20 mM glucose and then washed with methionine- and cysteine-free DMEM. Cells were labeled by adding 100 µCi of Redivue PRO-MIX (Amersham Biosciences) for 2 h and then incubated in DMEM containing 0.3 mM or 20 mM glucose.
For immunoprecipitation, anti-HA-agarose (Roche) was used. Immunoblotting was performed as described previously (17). To detect endogenous MafA protein, anti-c-Maf (M-153; Santa Cruz Biotechnology) was used because it cross-reacts with large Maf subfamily members (c-Maf, MafB, and MafA). It is referred to as anti-pan-Maf serum in this study. Anti-v-Maf (18) was used to detect MafABsu36I. The other primary antisera used in this study were as follows: anti-HA (MBL), anti-phospho-p44/42 mitogen-activated protein (MAP) kinase (Erk1/2) (Thr202/Tyr204) (Cell Signaling), anti-GSK3 (0011-A; Santa Cruz Biotechnology), anti-phospho-GSK3ß (Ser9) (Cell Signaling), anti-Akt (Cell Signaling), anti-phospho-Akt (Ser473) (Cell Signaling), anti-Gal4 (DBD) (Santa Cruz Biotechnology), anti-GFP (Clontech), and anti-TF-IID (TBP SI-1; Santa Cruz Biotechnology).
In vitro phosphorylation. To express recombinant HA-tagged MafA (rec-HA-MafA) in Escherichia coli, a DNA fragment containing HA-m-mafA was excised from pHygEF2/HA-m-mafA by digestion with NcoI (located at the initiator methionine) and EcoRI (located in the vector sequence downstream of the open reading frame) and inserted into the NcoI and XhoI sites of pET-14b (Novagen). The resultant plasmid, pET14b/HA-m-mafA, encoded rec-HA-MafA that lacked the amino-terminal His tag but contained its own His cluster, thus enabling one-step purification using Ni-nitrilotriacetic acid agarose (QIAGEN) under urea-denatured conditions. Purified rec-HA-MafA was dialyzed against HGKEDN buffer (20 mM HEPES, pH 7.9, 10% glycerol, 100 mM KCl, 0.2 mM EDTA, 1 mM dithiothreitol, 0.1% NP-40).
For the in vitro kinase assay, 2 ng of rec-HA-MafA and 1 µg of MIN6 nuclear extract or recombinant GSK3ß (Calbiochem) were combined in reaction buffer (final concentration of 5 mM HEPES, pH 7.9, 100 mM NaCl, 5 mM KCl, 0.8 mM dithiothreitol, 4.5% glycerol, 1 mM MgCl2, 10 mM Na3VO4, and 2.5 mM ATP) and incubated at 30°C for 60 min.
Luciferase assay. In1024 cells grown in 24-well plates were transfected with a total of 0.8 µg of plasmid DNA (0.1 µg luciferase plasmid, a total of 0.6 µg expression plasmids, and 0.1 µg pEF-Rluc) using 2 µl of Lipofectamine 2000 reagent (Invitrogen). Cells were harvested 24 h after transfection. Firefly and Renilla luciferase activities were measured using a dual luciferase assay system (Promega). Data represent the averages ± standard errors of three independent experiments.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We then screened several other types of kinase inhibitors in MIN6 cells for their abilities to alter the mobility of MafA during SDS-PAGE and found that SB216763, a specific inhibitor of GSK3, caused the rapidly migrating form of MafA to accumulate in a dose-dependent manner (Fig. 1D). Another GSK3 inhibitor, LiCl, had a similar effect (Fig. 1D). Treatment of other mouse (ßTC6 and NIT-1) and hamster (In1024) ß-cell lines with SB216763 also resulted in similar changes in MafA mobility (Fig. 1E). These results suggest that MafA is phosphorylated by GSK3 in ß cells.
Identification of conserved Ser and Thr residues at the amino terminus of MafA.
To determine the residues of MafA that were phosphorylated in ß cells, expression vectors encoding HA fused to full-length MafA (HA-MafA) or an amino-terminal truncation of MafA (MafABsu36I) were transiently transfected into In1024 cells, and MafA mobility in these cells in the presence of SB216763 was examined. The change in mobility of HA-MafA following SB216763 treatment was similar to that of endogenous MafA, whereas the mobility of MafABsu36I was unaffected (Fig. 2A). These results indicate that the GSK3 inhibitor-sensitive site(s) of phosphorylation of MafA is located in the amino-terminal region of MafA.
To identify the phosphorylated amino acid residue(s) in the amino-terminal region of MafA, we generated a set of substitution mutants of MafA, in which each Ser or Thr residue in this region was replaced with Ala, and examined the mobilities of the point mutants by SDS-PAGE. Mutation of Ser49, Thr53, Thr57, Ser61, or Ser65 (S49A, T53A, T57A, S61A, or S65A, respectively) increased MafA mobility (Fig. 2B, top). We also found that Ser72 and Ser342 of MafA were phosphorylated but that GSK3 was not involved in the phosphorylation of these residues (data not shown). In the background of a Ser72/Ser342 double substitution mutant, the mobility changes of S49A, T53A, T57A, S61A, and S65A mutants were more evident (Fig. 2B, bottom). In this set of experiments, we used the hamster insulinoma-derived cell line In1024 because of its high transfection efficiency compared to those of the other mouse ß-cell lines (MIN6, ßTC6, and NIT-1). We observed essentially the same mobility patterns for the Ala substitution mutants in MIN6 and NIT-1 cells (data not shown).
Attempts to identify the phosphorylation sites of MafA by mass spectrometry analysis were unsuccessful, most likely due to technical difficulties associated with detecting peptides containing multiple phosphoserine and phosphothreonine residues. However, the putative phosphorylation sites that we identified in the amino terminus of MafA (Ser49, Thr53, Thr57, Ser61, and Ser65) are well conserved throughout evolution among Maf family members and are spaced every 4 amino acids (Fig. 2C). It has been shown previously that phosphorylation by GSK3 requires prior phosphorylation of a Ser or Thr residue 4 residues carboxy terminal to the target site by another kinase (priming kinase) and that GSK3 sequentially phosphorylates every fourth Ser or Thr residue amino terminal to the priming site (7). Therefore, it seemed plausible that Ser65 of MafA functions as a priming site and is phosphorylated by an as-yet-unidentified priming kinase and that GSK3 sequentially phosphorylates Ser61, Thr57, Thr53, and Ser49. Consistent with this hypothesis, we observed that there were differences in the mobilities of the mutants (Fig. 2B). For example, the mobility of S65A was greater than that of S61A, most likely because Ser49, Thr53, Thr57, and Ser61, in addition to Ser65, were not phosphorylated in the S65A mutant, whereas at least at Ser65 was phosphorylated in the S61A mutant.
To test this hypothesis, we constructed MafA mutants with multiple mutations at each of the putative GSK3 phosphorylation sites and compared their mobilities. The mobility of a single substitution mutant was similar to that of a mutant with multiple substitutions at more-amino-terminal residues (Fig. 2D, top). For example, the mobility of the S61A mutant was similar to that of the 4A mutant (S49A T53A T57A S61A). This behavior of the mutants was more clearly evident when Ser72 and Ser342 were also mutated (Fig. 2D, bottom).
We also examined the effect of a GSK3 inhibitor on the mobility of the MafA phosphorylation site mutants. Treatment of cells with a GSK3 inhibitor altered the mobilities of WT MafA and the S49A, T53A, and T57A mutants to a form that appeared similar to that of the S61A mutant, whereas the mobilities of the S61A and S65A mutants were not affected (Fig. 2E). These results provide further evidence that Ser65 is the priming site and that Ser61, The57, Thr53, and Ser49 are then subsequently phosphorylated by GSK3.
Phosphorylation of MafA by GSK3 requires priming phosphorylation. Obtaining direct evidence of MafA phosphorylation by GSK3, by methods such as an in vitro kinase assay using recombinant MafA and GSK3, is not possible, since the priming kinase that phosphorylates Ser65 is unknown. To overcome this limitation, we performed an in vitro kinase assay using nuclear extracts prepared from MIN6 cells.
Full-length rec-HA-MafA was purified from bacteria and subjected to an in vitro kinase reaction using MIN6 nuclear extract. The mobility of purified rec-HA-MafA was similar to that of CIAP-treated HA-MafA expressed in MIN6 cells (Fig. 3A, lanes 2 and 3, form "U"). After the in vitro kinase reaction, a slower-migrating form of MafA that had a mobility similar to that of the hyperphosphorylated forms of HA-MafA in MIN6 cells (Fig. 3A, lane 1, form "F") appeared (Fig. 3A, lane 4). Thus, the results of the in vitro kinase reaction mimicked conditions observed in vivo. The addition of recombinant GSK3ß instead of MIN6 nuclear extract into the reaction mixture had no effect on MafA mobility (Fig. 3A, lane 6).
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To confirm the results of the in vitro kinase reaction, we treated MIN6 cells that stably expressed HA-MafA with the GSK3 inhibitor LiCl, and the "primed" form of MafA was purified by immunoprecipitation with an anti-HA antibody. The mobility of MafA was faster when the cells were treated with LiCl (Fig. 3C, top, lanes 1 and 2), similar to previous results (Fig. 1D). Following in vitro phosphorylation with recombinant GSK3ß, the slower-migrating forms of MafA were apparent (Fig. 3C, top, lane 3). When the kinase reaction was performed in the presence of [
-32P]ATP, 32P was incorporated into MafA (Fig. 3C, bottom). These results indicate that GSK3-mediated phosphorylation of MafA is dependent on a prior priming phosphorylation event.
Phosphorylated forms of MafA are unstable. Previously, it was shown that Erk phosphorylates serine and threonine residues of avian MafA which correspond to Ser14, Thr53, and Ser65 of mammalian MafA and that phosphorylation by Erk enhanced proteasome-dependent degradation of MafA in cultured lens cells (32). To examine the relationship between MafA protein stability and phosphorylation status in ß cells, we treated ßTC6 cells with the proteasome inhibitor MG132. In the presence of MG132, there was an accumulation of endogenous MafA protein (Fig. 4A). Similar results were observed for MIN6 cells that expressed exogenous MafA following treatment with MG132 or another proteasome inhibitor, lactacystin (Fig. 4B). Accumulation of exogenously expressed HA-MafA upon MG132 treatment was also observed in the ß-cell lines ßTC6 and NIT-1 but not in NIH 3T3 fibroblasts (Fig. 4C). These results together suggest that MafA is degraded via the proteasome pathway in a cell-type-specific manner.
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We next examined the transactivation activities of the MafA phosphorylation site mutants. A luciferase reporter gene driven by the human insulin promoter was cotransfected into In1024 cells with an expression plasmid encoding WT MafA or the 4A or 5A mutant of MafA. MafA alone was a weak activator of the insulin promoter, probably due to the high basal activity of the insulin promoter in the insulinoma-derived cell line, and the 4A and 5A mutants had comparable transactivation activities (Fig. 4F). Coexpression of Pdx1 and Beta2 had a synergistic effect on MafA activity (Fig. 4F), which was in good agreement with previous observations (2, 5, 16, 55). Both the 4A and 5A mutants also functioned synergistically with Pdx1 and Beta2 and exhibited higher luciferase activities than WT MafA. However, the expression levels of the 4A and 5A mutants were higher than that of WT MafA (Fig. 4F, inset); thus, additional experiments are needed to quantitate the relative intrinsic transactivation potentials of MafA and the mutants. At least, these results indicate that amino-terminal phosphorylation is not essential for transactivation by MafA.
To determine whether the amino-terminal region of MafA was sufficient to trigger both phosphorylation and degradation, this region was fused with the DBD of Gal4 (Gal4-MafA-WT) (Fig. 5A). This system also enabled us to examine the transactivation activity of the amino terminus of MafA, as this region has been shown to contain a transactivator domain (15, 35). The corresponding regions of the 4A and 5A mutants were also fused to Gal4-DBD to create Gal4-MafA-4A and Gal4-MafA-5A, respectively. The fusion proteins were expressed in In1024 cells, and cells were treated with SB216763 or MG132. Gal4-MafA-WT was phosphorylated by GSK3 and sensitive to MG132, whereas Gal4-MafA-4A and -5A were not phosphorylated by GSK3 and were less sensitive to MG132 (Fig. 5B). Analysis of CHX treatment showed that both Gal4-MafA-4A and -5A also degraded more slowly than Gal4-MafA-WT (Fig. 5C). All three proteins exhibited significant transcriptional activities, which correlated with their expression levels (Fig. 5D). These results suggest that the amino-terminal region of MafA contains the domains that are sufficient for transactivation as well as phosphorylation-dependent degradation.
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To confirm these results, we established MIN6 clonal cell lines that stably expressed wild-type MafA or the 4A mutant fused to IRES-EGFP under the control of the constitutively active EF-1
promoter (Fig. 6E, top). The expression of exogenous wild-type MafA was comparable to that of endogenous MafA (Fig. 6F), and exogenously expressed MafA protein accumulated in response to glucose (Fig. 6E, bottom). In contrast, the 4A mutant was more abundant than endogenous MafA (Fig. 6F) and was insensitive to glucose (Fig. 6E, bottom).
Exogenously expressed MafA and the 4A and 5A mutants accumulated to similar levels under both low- and high-glucose conditions in NIH 3T3 (mouse embryonic fibroblast), BHK21 (Syrian hamster kidney), and CHO (Chinese hamster ovary) cells, (Fig. 6G), which suggests that phosphorylation-dependent regulation of MafA stability in response to glucose is specific to ß cells.
MafA degradation rate, but not phosphorylation, is regulated by glucose. Previous studies have shown that GSK3 activity is negatively regulated by Akt-mediated phosphorylation (4) and that Akt is activated by glucose in ß cells (46). We hypothesized that glucose inhibited GSK3 activity through Akt, leading to MafA hypophosphorylation and accumulation. The levels of phosphorylated Akt (active form) and phosphorylated GSK3ß (inactive form) both increased under high- glucose conditions in MIN6 cells (Fig. 7A). However, the phosphorylation status of MafA appeared to be unchanged by glucose (Fig. 7A; see also Fig. 6A and B).
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We also examined MafA kinase activity in MIN6 nuclear extracts in vitro. Nuclear extracts were prepared from MIN6 cells grown in low- or high-glucose medium, and an in vitro phosphorylation reaction was carried out using recombinant MafA protein as a substrate. The efficiencies of phosphorylation of MafA were similar for nuclear extracts from cells grown under both conditions (Fig. 7D). These results suggest that while glucose stimulates the Akt-GSK3 pathway in MIN6 cells, MafA phosphorylation activity is unchanged by glucose.
We next examined the degradation rates of MafA under low- and high-glucose conditions. MIN6 cells stably expressing HA-MafA-WT were labeled with [35S]methionine and [35S]cysteine for 2 h in low- or high-glucose medium, and then the medium was changed to low- or high-glucose medium. Cell extracts were subjected to immunoprecipitation, followed by SDS-PAGE and autoradiography. As seen in Fig. 8A, 35S-labeled HA-MafA disappeared more rapidly under low-glucose conditions than under high-glucose conditions.
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When we examined the effect of a GSK3 inhibitor on the half-life of endogenous MafA, we found that the rate of MafA degradation was decreased both in the presence and in the absence of glucose (Fig. 8C). These results provide further evidence that phosphorylation by GSK3 is required for the rapid degradation of MafA and that the degradation rate, not the phosphorylation status, of MafA protein is regulated by glucose.
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DISCUSSION |
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Our data suggest that MafA is phosphorylated in ß-cell lines at seven Ser and Thr residues (Ser49, Thr53, Thr57, Ser61, Ser65, Ser72, and Ser342) (summarized in Fig. 2C). Previous studies have shown that chicken and quail homologues of MafA are phosphorylated by Erk at Ser14, Thr57, and Ser65 in vitro and in vivo (in primary lens, primary neural retina, and HeLa cells) (3, 32, 33). It was also shown previously that Thr57, as well as Thr113 and Ser272 (equivalent to Thr131 and Ser342 of m-MafA, respectively), is phosphorylated by p38 MAP kinase in vitro and in 293T cells (45). While these phosphoacceptor sites are conserved in several species (Fig. 2C), treatment of ß cells with pharmacological inhibitors of the MAP kinases Erk, p38, and Jun N-terminal protein kinase, either alone or in combination, had no effect on the mobility of endogenous MafA during SDS-PAGE (Fig. 1C and data not shown), indicating that the contribution of MAP kinases to MafA phosphorylation in ß cells is insignificant. Our data suggest that in ß cells, GSK3 phosphorylates Ser49, Thr53, Thr57, and Ser61 of MafA (Fig. 1 and 2). Ser65 appeared to be phosphorylated by an unknown kinase and to serve as a priming site for subsequent phosphorylation at additional sites by GSK3. It should be noted that GSK3 phosphorylates MafA without a priming phosphorylation event with low efficiency in ß cells. For example, a small fraction of the S65A mutant appeared to be fully phosphorylated and was sensitive to the GSK3 inhibitor (Fig. 2D and E). The candidate phosphorylation sites that were identified in the current study, Thr53, Thr57, and Ser65, as well as the other phosphorylated residues of MafA in ß cells (Ser72 and Ser342) or in other systems (Ser14 and Thr131), are followed by a Pro residue. Thus, they can potentially serve as phosphoacceptor sites for proline- directed kinases, including MAP kinases, and may be phosphorylated by different kinases depending on cell type. The kinase(s) responsible for phosphorylation of Ser65, Ser72, and Ser342 in ß cells has yet to be identified.
We also present evidence that GSK3-phosphorylated MafA undergoes rapid degradation in ß cells. This is consistent with previous findings demonstrating that mutation of chicken L-Maf at sites of Erk phosphorylation (Thr57 and Ser65) resulted in significant accumulation of L-Maf in primary lens cells (32). Many proteins, including transcription factors, are reported substrates of GSK3, and one of the functional consequences of GSK3 phosphorylation is degradation of the target protein. For example, phosphorylation of ß-catenin, c-Myc, and c-Jun targets these proteins for polyubiquitination by specific E3 ligases and degradation by the proteasome (10, 22, 50, 51). We show here that phosphorylated forms of MafA in ß cells accumulate in response to treatment with the proteasome inhibitors MG132 and lactacystin, which suggests that phosphorylated MafA is also degraded by the proteasomal pathway.
MafA has been identified as a trans-acting factor that binds to the glucose-responsive cis-element RIPE3b/C1, and glucose-dependent accumulation of MafA in ß cells has been demonstrated previously (15, 17, 55). We and others showed previously that both mafA mRNA and MafA protein levels increase in response to glucose (11, 17, 49), which indicates that glucose regulates MafA at least at both the transcriptional and posttranscriptional levels. Based on our current observation that glucose-dependent regulation of MafA was almost lost when degradation was inhibited by MG132, we propose that glucose regulates MafA mainly by controlling protein stability. Additional studies are needed to determine the relative contributions of transcriptional and posttranslational regulation. Harmon et al. (13) showed that MafA protein loss by chronic exposure to oxidative stress is mediated at the level of protein stability. In the current study, we show that pharmacological inhibition of GSK3 in MIN6 cells results in an almost complete loss of MafA regulation by glucose and that phosphorylation-deficient mutants of MafA are very stable, even under low-glucose conditions. These results suggest that glucose-mediated regulation of MafA requires phosphorylation of MafA by GSK3 and that GSK3 and an as-yet-unidentified priming kinase are the major kinases in ß cells that affect MafA stability. It is of note that the GSK3 inhibitor affected MafA abundance even in the presence of glucose (Fig. 6B). The reason for this is not clear, but it is possible that GSK3 inhibition may stimulate translation by affecting inhibitory phosphorylation of the translation initiation factor eIF2B (52).
We demonstrated that the overall phosphorylation status of MafA and the MafA-directed kinase activity in MIN6 cells are unchanged by glucose. Therefore, we propose that MafA is constitutively phosphorylated in ß cells regardless of glucose concentration. We found that the half-life of MafA was prolonged by glucose in ß cells. Exogenously expressed MafA protein was phosphorylated similarly in other types of cells, such as NIH 3T3, BHK21, and CHO cells, but MafA protein abundance was not regulated by glucose. This result is contradictory to a report by Kajihara et al. in which MafA abundance was regulated by glucose concentration even in NIH 3T3 cells (15). The reason for this discrepancy is not known. From our observations, we speculate that glucose-dependent degradation of MafA is cell type specific. The precise molecular mechanism(s) of regulation of the degradation of MafA in response to glucose is unknown. An as-yet-unidentified phospho-MafA-specific E3 ubiquitin ligase might be expressed specifically in ß cells and may be regulated by glucose concentration. Recently, Vanderford et al. reported that glucose induces MafA protein expression via the hexoseamine biosynthetic pathway and O-linked glycosylation (49). The putative phospho-MafA-specific E3 ubiquitin ligase might be a target of this pathway. Our analysis of changes in the mobility of MafA during SDS-PAGE does not exclude the possibility that undetectable levels of phosphorylation or other modifications of MafA account for its selective degradation under low-glucose conditions in ß cells. We have examined the O-glycosylation status of MafA by using an anti-O-GlcNAc antibody and have found no evidence for O glycosylation of MafA (unpublished observation).
MafA plays an important role in insulin gene transcription and ß-cell function and is a promising therapeutic target for ß-cell regeneration and differentiation. Therefore, identification of the molecular components of MafA degradation and their function is very important for clarifying the link between MafA and glucose sensing in ß cells and for understanding ß-cell biology and the pathogenesis of diabetes.
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ACKNOWLEDGMENTS |
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This work was supported by Grants-in-Aid for Scientific Research on Priority Area, for Encouragement of Young Scientists, and for the 21st Century COE Research from the Ministry of Education, Culture, Sports, Science and Technology, a grant from Foundation for Nara Institute of Science and Technology, and a grant from the Sagawa Foundation for Promotion of Cancer Research to K.K. Song-iee Han was supported by a Postdoctoral Fellowship for Foreign Researchers from the Japan Society for the Promotion of Science.
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FOOTNOTES |
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Published ahead of print on 6 August 2007.
Present address: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki 305-8572, Japan.
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REFERENCES |
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2. Aramata, S., S. I. Han, K. Yasuda, and K. Kataoka. 2005. Synergistic activation of the insulin gene promoter by the beta-cell enriched transcription factors MafA, Beta2, and Pdx1. Biochim. Biophys. Acta 1730:41-46.[Medline]
3. Benkhelifa, S., S. Provot, E. Nabais, A. Eychene, G. Calothy, and M. P. Felder-Schmittbuhl. 2001. Phosphorylation of MafA is essential for its transcriptional and biological properties. Mol. Cell. Biol. 21:4441-4452.
4. Cross, D. A., D. R. Alessi, P. Cohen, M. Andjelkovich, and B. A. Hemmings. 1995. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378:785-789.[CrossRef][Medline]
5. Docherty, H. M., C. W. Hay, L. A. Ferguson, J. Barrow, E. Durward, and K. Docherty. 2005. Relative contribution of PDX-1, MafA and E47/beta2 to the regulation of the human insulin promoter. Biochem. J. 389:813-820.[CrossRef][Medline]
6. Elrick, L. J., and K. Docherty. 2001. Phosphorylation-dependent nucleocytoplasmic shuttling of pancreatic duodenal homeobox-1. Diabetes 50:2244-2252.
7. Fiol, C. J., A. M. Mahrenholz, Y. Wang, R. W. Roeske, and P. J. Roach. 1987. Formation of protein kinase recognition sites by covalent modification of the substrate. Molecular mechanism for the synergistic action of casein kinase II and glycogen synthase kinase 3. J. Biol. Chem. 262:14042-14048.
8. German, M. S., and J. Wang. 1994. The insulin gene contains multiple transcriptional elements that respond to glucose. Mol. Cell. Biol. 14:4067-4075.
9. Gotoh, M., H. Ohzato, J. Porter, T. Maki, and A. P. Monaco. 1990. Crucial role of pancreatic ductal collagenase injection for isolation of pancreatic islets. Horm. Metab. Res. Suppl. 25:10-16.[Medline]
10. Gregory, M. A., Y. Qi, and S. R. Hann. 2003. Phosphorylation by glycogen synthase kinase-3 controls c-myc proteolysis and subnuclear localization. J. Biol. Chem. 278:51606-51612.
11. Hagman, D. K., L. B. Hays, S. D. Parazzoli, and V. Poitout. 2005. Palmitate inhibits insulin gene expression by altering PDX-1 nuclear localization and reducing MafA expression in isolated rat islets of Langerhans. J. Biol. Chem. 280:32413-32418.
12. Hani, E. H., D. A. Stoffers, J. C. Chevre, E. Durand, V. Stanojevic, C. Dina, J. F. Habener, and P. Froguel. 1999. Defective mutations in the insulin promoter factor-1 (IPF-1) gene in late-onset type 2 diabetes mellitus. J. Clin. Investig. 104:R41-R48.[Medline]
13. Harmon, J. S., R. Stein, and R. P. Robertson. 2005. Oxidative stress-mediated, post-translational loss of MafA protein as a contributing mechanism to loss of insulin gene expression in glucotoxic beta cells. J. Biol. Chem. 280:11107-11113.
14. Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51-59.[CrossRef][Medline]
15. Kajihara, M., H. Sone, M. Amemiya, Y. Katoh, M. Isogai, H. Shimano, N. Yamada, and S. Takahashi. 2003. Mouse MafA, homologue of zebrafish somite Maf1, contributes to the specific transcriptional activity through the insulin promoter. Biochem. Biophys. Res. Commun. 312:831-842.[CrossRef][Medline]
16. Kaneto, H., T. A. Matsuoka, Y. Nakatani, T. Miyatsuka, M. Matsuhisa, M. Hori, and Y. Yamasaki. 2005. A crucial role of MafA as a novel therapeutic target for diabetes. J. Biol. Chem. 280:15047-15052.
17. Kataoka, K., S. I. Han, S. Shioda, M. Hirai, M. Nishizawa, and H. Handa. 2002. MafA is a glucose-regulated and pancreatic beta-cell-specific transcriptional activator for the insulin gene. J. Biol. Chem. 277:49903-49910.
18. Kataoka, K., M. Nishizawa, and S. Kawai. 1993. Structure-function analysis of the maf oncogene product, a member of the b-Zip protein family. J. Virol. 67:2133-2141.
19. Kataoka, K., S. Shioda, K. Yoshitomo-Nakagawa, H. Handa, and M. Nishizawa. 2001. Maf and Jun nuclear oncoproteins share downstream target genes for inducing cell transformation. J. Biol. Chem. 276:36849-36856.
20. Kataoka, K., K. Yoshitomo-Nakagawa, S. Shioda, and M. Nishizawa. 2001. A set of Hox proteins interact with the Maf oncoprotein to inhibit its DNA binding, transactivation, and transforming activities. J. Biol. Chem. 276:819-826.
21. Le Lay, J., T. A. Matsuoka, E. Henderson, and R. Stein. 2004. Identification of a novel PDX-1 binding site in the human insulin gene enhancer. J. Biol. Chem. 279:22228-22235.
22. Liu, C., Y. Li, M. Semenov, C. Han, G. H. Baeg, Y. Tan, Z. Zhang, X. Lin, and X. He. 2002. Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 108:837-847.[CrossRef][Medline]
23. Macfarlane, W. M., T. M. Frayling, S. Ellard, J. C. Evans, L. I. Allen, M. P. Bulman, S. Ayres, M. Shepherd, P. Clark, A. Millward, A. Demaine, T. Wilkin, K. Docherty, and A. T. Hattersley. 1999. Missense mutations in the insulin promoter factor-1 gene predispose to type 2 diabetes. J. Clin. Investig. 104:R33-R39.[Medline]
24. Macfarlane, W. M., C. M. McKinnon, Z. A. Felton-Edkins, H. Cragg, R. F. James, and K. Docherty. 1999. Glucose stimulates translocation of the homeodomain transcription factor PDX1 from the cytoplasm to the nucleus in pancreatic beta-cells. J. Biol. Chem. 274:1011-1016.
25. MacFarlane, W. M., M. L. Read, M. Gilligan, I. Bujalska, and K. Docherty. 1994. Glucose modulates the binding activity of the beta-cell transcription factor IUF1 in a phosphorylation-dependent manner. Biochem. J. 303:625-631.[Medline]
26. Malecki, M. T., U. S. Jhala, A. Antonellis, L. Fields, A. Doria, T. Orban, M. Saad, J. H. Warram, M. Montminy, and A. S. Krolewski. 1999. Mutations in NEUROD1 are associated with the development of type 2 diabetes mellitus. Nat. Genet. 23:323-328.[CrossRef][Medline]
27. Marshak, S., H. Totary, E. Cerasi, and D. Melloul. 1996. Purification of the beta-cell glucose-sensitive factor that transactivates the insulin gene differentially in normal and transformed islet cells. Proc. Natl. Acad. Sci. USA 93:15057-15062.
28. Matsuoka, T. A., L. Zhao, I. Artner, H. W. Jarrett, D. Friedman, A. Means, and R. Stein. 2003. Members of the large Maf transcription family regulate insulin gene transcription in islet beta cells. Mol. Cell. Biol. 23:6049-6062.
29. Miyazaki, J., K. Araki, E. Yamato, H. Ikegami, T. Asano, Y. Shibasaki, Y. Oka, and K. Yamamura. 1990. Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology 127:126-132.[Abstract]
30. Naya, F. J., H. P. Huang, Y. Qiu, H. Mutoh, F. J. DeMayo, A. B. Leiter, and M. J. Tsai. 1997. Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/neuroD-deficient mice. Genes Dev. 11:2323-2334.
31. Naya, F. J., C. M. Stellrecht, and M. J. Tsai. 1995. Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor. Genes Dev. 9:1009-1019.
32. Ochi, H., H. Ogino, Y. Kageyama, and K. Yasuda. 2003. The stability of the lens-specific Maf protein is regulated by fibroblast growth factor (FGF)/ERK signaling in lens fiber differentiation. J. Biol. Chem. 278:537-544.
33. Ochi, H., K. Sakagami, A. Ishii, N. Morita, M. Nishiuchi, H. Ogino, and K. Yasuda. 2004. Temporal expression of L-Maf and RaxL in developing chicken retina are arranged into mosaic pattern. Gene Expr. Patterns 4:489-494.[CrossRef][Medline]
34. Ohlsson, H., K. Karlsson, and T. Edlund. 1993. IPF1, a homeodomain-containing transactivator of the insulin gene. EMBO J. 12:4251-4259.[Medline]
35. Olbrot, M., J. Rud, L. G. Moss, and A. Sharma. 2002. Identification of beta-cell-specific insulin gene transcription factor RIPE3b1 as mammalian MafA. Proc. Natl. Acad. Sci. USA 99:6737-6742.
36. Petersen, H. V., J. N. Jensen, R. Stein, and P. Serup. 2002. Glucose induced MAPK signalling influences NeuroD1-mediated activation and nuclear localization. FEBS Lett. 528:241-245.[CrossRef][Medline]
37. Petersen, H. V., M. Peshavaria, A. A. Pedersen, J. Philippe, R. Stein, O. D. Madsen, and P. Serup. 1998. Glucose stimulates the activation domain potential of the PDX-1 homeodomain transcription factor. FEBS Lett. 431:362-366.[CrossRef][Medline]
38. Petersen, H. V., P. Serup, J. Leonard, B. K. Michelsen, and O. D. Madsen. 1994. Transcriptional regulation of the human insulin gene is dependent on the homeodomain protein STF1/IPF1 acting through the CT boxes. Proc. Natl. Acad. Sci. USA 91:10465-10469.
39. Poitout, V., D. Hagman, R. Stein, I. Artner, R. P. Robertson, and J. S. Harmon. 2006. Regulation of the insulin gene by glucose and fatty acids. J. Nutr. 136:873-876.
40. Rafiq, I., G. da Silva Xavier, S. Hooper, and G. A. Rutter. 2000. Glucose-stimulated preproinsulin gene expression and nuclear trans-location of pancreatic duodenum homeobox-1 require activation of phosphatidylinositol 3-kinase but not p38 MAPK/SAPK2. J. Biol. Chem. 275:15977-15984.
41. Schreiber, E., P. Matthias, M. M. Muller, and W. Schaffner. 1989. Rapid detection of octamer binding proteins with ‘mini-extracts’, prepared from a small number of cells. Nucleic Acids Res. 17:6419.
42. Sharma, A., D. Fusco-DeMane, E. Henderson, S. Efrat, and R. Stein. 1995. The role of the insulin control element and RIPE3b1 activators in glucose-stimulated transcription of the insulin gene. Mol. Endocrinol. 9:1468-1476.[Abstract]
43. Sharma, A., and R. Stein. 1994. Glucose-induced transcription of the insulin gene is mediated by factors required for beta-cell-type-specific expression. Mol. Cell. Biol. 14:871-879.
44. Shieh, S. Y., and M. J. Tsai. 1991. Cell-specific and ubiquitous factors are responsible for the enhancer activity of the rat insulin II gene. J. Biol. Chem. 266:16708-16714.
45. Sii-Felice, K., C. Pouponnot, S. Gillet, L. Lecoin, J. A. Girault, A. Eychene, and M. P. Felder-Schmittbuhl. 2005. MafA transcription factor is phosphorylated by p38 MAP kinase. FEBS Lett. 579:3547-3554.[CrossRef][Medline]
46. Srinivasan, S., E. Bernal-Mizrachi, M. Ohsugi, and M. A. Permutt. 2002. Glucose promotes pancreatic islet beta-cell survival through a PI 3-kinase/Akt-signaling pathway. Am. J. Physiol. Endocrinol. Metab. 283:E784-E793.
47. Stoffers, D. A., J. Ferrer, W. L. Clarke, and J. F. Habener. 1997. Early-onset type-II diabetes mellitus (MODY4) linked to IPF1. Nat. Genet. 17:138-139.[CrossRef][Medline]
48. Uchida, S., S. Watanabe, T. Aizawa, A. Furuno, and T. Muto. 1979. Polyoncogenicity and insulinoma-inducing ability of BK virus, a human papovavirus, in Syrian golden hamsters. J. Natl. Cancer Inst. 63:119-126.[Medline]
49. Vanderford, N. L., S. S. Andrali, and S. Ozcan. 2007. Glucose induces MafA expression in pancreatic beta cell lines via the hexosamine biosynthetic pathway. J. Biol. Chem. 282:1577-1584.
50. Wei, W., J. Jin, S. Schlisio, J. W. Harper, and W. G. Kaelin, Jr. 2005. The v-Jun point mutation allows c-Jun to escape GSK3-dependent recognition and destruction by the Fbw7 ubiquitin ligase. Cancer Cell 8:25-33.[CrossRef][Medline]
51. Welcker, M., A. Orian, J. Jin, J. E. Grim, J. W. Harper, R. N. Eisenman, and B. E. Clurman. 2004. The Fbw7 tumor suppressor regulates glycogen synthase kinase 3 phosphorylation-dependent c-Myc protein degradation. Proc. Natl. Acad. Sci. USA 101:9085-9090.
52. Welsh, G. I., C. M. Miller, A. J. Loughlin, N. T. Price, and C. G. Proud. 1998. Regulation of eukaryotic initiation factor eIF2B: glycogen synthase kinase-3 phosphorylates a conserved serine which undergoes dephosphorylation in response to insulin. FEBS Lett. 421:125-130.[CrossRef][Medline]
53. Wilson, M. E., D. Scheel, and M. S. German. 2003. Gene expression cascades in pancreatic development. Mech. Dev. 120:65-80.[CrossRef][Medline]
54. Zhang, C., T. Moriguchi, M. Kajihara, R. Esaki, A. Harada, H. Shimohata, H. Oishi, M. Hamada, N. Morito, K. Hasegawa, T. Kudo, J. D. Engel, M. Yamamoto, and S. Takahashi. 2005. MafA is a key regulator of glucose-stimulated insulin secretion. Mol. Cell. Biol. 25:4969-4976.
55. Zhao, L., M. Guo, T. A. Matsuoka, D. K. Hagman, S. D. Parazzoli, V. Poitout, and R. Stein. 2005. The islet beta cell-enriched MafA activator is a key regulator of insulin gene transcription. J. Biol. Chem. 280:11887-11894.
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