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Biosignal Research Center, Kobe University, Kobe 657-8501, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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-subunit and the phosphorylation of the catalytic 
-subunit at Thr172 by AMPK kinases such as LKB1 and Ca2+/calmodulin-dependent protein kinase kinases (Kemp et al. 2003; Towler & Hardie 2007). The AMPK activity is elevated in the cells treated with 2-deoxyglucose (2-DG), a non-metabolizable glucose analog that increases the intracellular AMP:ATP ratio, and is attenuated by Compound C, a potent inhibitor of this protein kinase. Thus, mTOR and AMPK are essential in the regulation of cellular activities as major sensors of nutrient availability. Interestingly, AMPK directly phosphorylates TSC2 and raptor to suppress the mTOR signaling pathway (Inoki et al. 2003; Gwinn et al. 2008). Therefore, AMPK and mTOR are not independent, but these two nutrient-dependent signaling mechanisms operate in a concerted manner. In addition to the protein phosphorylation reaction by these protein kinases, O-linked glycosylation, which uses UDP-N-acetylglucosamine (UDP-GlcNAc), a glucose metabolite in the hexosamine signaling pathway, is involved in the nutritional signaling mechanisms (Marshall 2006). Glycosylation is regarded to be restricted to extracellular and secreted proteins, however, various proteins in the cytoplasm and nucleus have been shown to be modified at serine and threonine residues using UDP-GlcNAc via an O-glycosidic bond (O-linked β-N-acetylglucosamine (O-GlcNAc)) (Hart 1997). The modification of the proteins with O-GlcNAc in response to cellular signals is reversible and abundant as protein phosphorylation, and has been attracted a great deal of attention (Zachara & Hart 2006). We have established the method for the identification of novel phosphoproteins by the combination of the immunoprecipitation using the phosphorylation site specific antibody and subsequent mass fingerprinting using mass spectrometry, and identified Golgi-specific brefeldin A resistance factor 1 (GBF1), a guanine nucleotide exchange factor for the ADP-ribosylation factor (Arf) family, as a phosphoprotein from the glucose-depleted cells (Miyamoto et al. 2008). In this study, glutamine : fructose-6-phosphate amidotransferase 1 (GFAT1) was isolated, which is the first and rate-limiting enzyme for the entry of glucose into the hexosamine signaling pathway to generate UDP-GlcNAc in mammals. The phosphorylation site of GFAT1 was identified to evaluate the role of the modification reaction for the regulation of its enzymatic activity.
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Results |
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The anti-phospho Akt substrates (PAS) antibody was used to screen the nutrient-regulated phosphoprotein as described previously (Miyamoto et al. 2008). Namely, CHO-IR cells, which are derived from Chinese hamster ovary, were cultured in the medium sufficient or deprived of nutrients, that is, amino acids and glucose, and the immunoprecipitates with the PAS antibody from the cells were subjected to SDS-PAGE and visualized by silver staining (Fig. 1A). Several protein bands were isolated by the PAS antibody from the nutrient-depleted and nutrient-replete cells, respectively (Fig. 1A, right two lanes), whereas no significant protein band was observed in the control lanes where the lysis buffer alone without the cell lysate was applied to immunoprecipitation by this antibody (Fig. 1A, lane L) and normal rabbit globulin was employed for immunoprecipitation (Fig. 1A, left lane). They included GBF1, a Golgi apparatus-associated guanine nucleotide exchange factor for the ARF family of small GTPase, and plectin, a linker protein that associates with the cytoskeletal filament networks and many membrane proteins, in the nutrient-depleted and -replete cells, respectively, as reported previously (Miyamoto et al. 2008). In addition to these proteins, a protein with an approximate molecular mass of 80 kDa, designated as p80, was recovered as a major protein from the nutrient-deprived cells. Protein identification by mass spectrometry assigned p80 as the isoform 1 of human GFAT (NCBI accession number NP_002047 [GenBank] ), which is the rate-limiting enzyme in the hexosamine signaling pathway (Kudlow 2006). GFAT has three domains: a glutamine amidotransferases class-II domain, which transfers amide nitrogen from glutamine to substrate, and two sugar isomerase domains, that are found as a phosphosugar-binding domain in phosphosugar isomerases and phosphosugar-binding proteins (Massiere & Badet-Denisot 1998; Bateman 1999; Teplyakov et al. 2001), and catalyzes the first step in the hexosamine signaling pathway by transferring the amino group from glutamine to fructose-6-phosphate producing glucosamine-6-phosphate and glutamic acid. GFAT comprises of two isoforms found in various tissues in human (McKnight et al. 1992; Oki et al. 1999): GFAT1 is abundantly expressed in the placenta, pancreas, and testis, whereas GFAT2, which shows 79% amino acid sequence identity to GFAT1, is expressed highly in the central nervous system. The peptide mass list obtained from twenty peptide fragments matched with GFAT1: four of them were common to both isoforms, and the other 16 fitted GFAT1 but none of them was specific to GFAT2. Thus, p80 was concluded to contain a hamster homologue of GFAT1. A possibility is not eliminated that this protein band includes GFAT2, but p80 seems to be mainly composed of GFAT1. The immunoblot analysis of the immunoprecipitates with the PAS antibody confirmed that p80 is GFAT, employing the antibody raised against the C-terminal polypeptide sequence of the protein, which is common to both of the human GFAT isoforms (Fig. 1B, top). Consistently, the PAS antibody recognized GFAT immunoprecipitated from the nutrient-depleted cells more efficiently than the protein recovered from the nutrient-replete cells (Fig. 1B, bottom). These results indicate that GFAT, practically GFAT1, is phosphorylated in response to nutrient depletion.
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The nutritional starvation-sensitive phosphorylation of endogenous GFAT implies that GFAT is phosphorylated in the downstream of AMPK as in the case of GBF1 (Miyamoto et al. 2008). Thus, the effect of glucose depletion was examined on the phosphorylation of endogenous GFAT employing acetyl-CoA carboxylase (ACC), a typical substrate protein of AMPK, as a positive control (Fig. 2A). As expected, the phosphorylation of endogenous GFAT was induced by glucose depletion and was potentiated by 2-DG, a compound that lowers the intracellular ATP concentration to induce AMPK activation by blocking the cellular glucose utilization, in a manner parallel to that of ACC. In contrast, the amino acids in the culture medium, that activate mTORC1, did not affect the phosphorylation of endogenous GFAT. To confirm whether GFAT1 is phosphorylated in the downstream of AMPK, the phosphorylation of FLAG-tagged human GFAT1 was examined in the transfected cells (Fig. 2B). As in the case of endogenous GFAT, the 2-DG treatment enhanced the phosphorylation of FLAG-GFAT1, and the incubation of the cells with Compound C, an AMPK inhibitor (Zhou et al. 2001), greatly reduced the 2-DG-induced phosphorylation of GFAT1. Furthermore, the role of AMPK was examined by expressing the constitutively active and kinase-negative mutants of AMPK in the cells with myc-GFAT1 (Fig. 2C). In the cells transfected with the wild-type AMPK [GST-AMPK (WT)], the phosphorylation of myc-GFAT1 was induced by the 2-DG treatment, and the 2-DG-induced phosphorylation of myc-GFAT1 was attenuated by the introduction of the kinase-negative mutant of AMPK [GST-AMPK (K45R)]. On the other hand, the phosphorylation of myc-GFAT1 was observed in the cells expressing the constitutively active mutant of AMPK [GST-AMPK (1-312)] irrespective of the absence of 2-DG. The effects of over-expression of the AMPK mutants on the phosphorylation of ACC were, however, not so evident in the lysate as immunoprecipitated FLAG-GFAT1, presumably because the transfection efficiencies of the AMPK mutants are not high in these cells. Taken together, it was concluded that GFAT1 is phosphorylated in the downstream of AMPK in the cells exposed to metabolic stress accompanying the decrease in intracellular ATP concentration.
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Then, the phosphorylation site in human GFAT1 which is recognized by the PAS antibody was examined. AMPK has the consensus recognition motif, 
-[β,X]-X-X-Ser/Thr-X-X-X-, where , β, and X are hydrophobic, basic, and any amino acid, respectively, and the residues in the bracket are in any order (Towler & Hardie 2007). The PAS antibody reacts with the motif sequence phosphorylated at Arg-X-Arg-X-X-Ser/Thr and Arg-X-X-Ser/Thr (Fujita et al. 2002; Miyamoto et al. 2008). The latter is compatible with the phosphorylation motif sequence of AMPK as described above. In human GFAT1, the sequence surrounding Ser243 exclusively fits with both of the motifs of AMPK and the PAS antibody, which is conserved among different species as aligned in Fig. 3A. Therefore, we produced the GFAT1 mutant replacing Ser243 by Ala and the phospho-specific rabbit polyclonal antibody for Ser243 (anti-pS243) based on human GFAT1 sequence to examine the phosphorylation of GFAT1 in intact cells. The 2-DG-induced phosphorylation at Ser243 in the wild-type GFAT1 was detected by the anti-pS243 antibody as in the case of the PAS antibody, and the mutation at Ser243 in GFAT1 vanished the immunoreaction by the anti-pS243 and PAS antibodies (Fig. 3B).
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-32P]ATP with the wild-type or kinase-negative AMPK obtained from the 2-DG-treated and control cells, respectively (Fig. 3C). FLAG-GFAT1 (WT) was significantly phosphorylated by the wild-type AMPK (WT) obtained from the 2-DG-treated HEK293 cells, but was not phosphorylated by the kinase-negative AMPK (K45R) (Fig. 3C, left panels). Furthermore, the phosphorylation of GFAT1 by AMPK obtained from 2-DG treated cells vanished when S243A was employed as a substrate (Fig. 3C, right panels). These results indicate that GFAT1 is directly phosphorylated at Ser243 by AMPK. Inhibition of GFAT1 activity by AMPK
To study the significance of the AMPK-mediated phosphorylation of GFAT1, the enzymatic activity of GFAT was examined using established glutamate dehydrogenase method (Marshall et al. 1991; Ye et al. 2004) (Fig. 4). The generation of glutamate, one of the products in the GFAT reaction, was assayed as the reduction of acetylpyridine adenine dinucleotide (APAD) to APADH by the glutamate dehydrogenase reaction with glutamate, which is determined directly by the increase of absorbance at 370 nm. CHO-IR cells were treated with 2-DG and Compound C, and the cell lysates were subjected to the GFAT1 assay. The phosphorylation of GFAT was evident in 2-DG treated cells, and this phosphorylation was attenuated by the preincubation of the cells with the AMPK inhibitor, Compound C (Fig. 4, bottom). Under these conditions, the 2-DG-treatment significantly inhibited the enzymatic activity of GFAT, and Compound C-preincubation partially prevented 2-DG-induced decrease in the GFAT activity (Fig. 4, top). These results indicate that the AMPK-mediated phosphorylation of GFAT reduces its enzymatic activity.
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Discussion |
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In this study, GFAT1, the rate-limiting enzyme in the UDP-GlcNAc synthesis pathway, was identified as a protein phosphorylated in the glucose-deprived cells by the screening of the proteins phosphorylated by the changes of the nutritional conditions employing the combination of the immunoprecipitation by the PAS antibody and subsequent protein identification by mass spectrometry. Product ion mass fingerprinting analysis identified peptide fragments specific to GFAT1. Following analysis revealed that the PAS antibody recognized GFAT1 phosphorylated at Ser243 by AMPK. Namely, GFAT1 was revealed to be a novel in vivo substrate of AMPK. This serine residue is conserved in GFAT2 as Ser244. The phosphorylation of ACC, the positive control of AMPK substrate, was induced by glucose starvation and was further enhanced by the 2-DG treatment employing the cell lysates, whereas the starvation-induced phosphorylation of GFAT was not facilitated significantly by this AMPK activator even the immunoprecipitates were used for the analysis (Fig. 2A). Thus, GFAT seems to be phosphorylated by AMPK more sensitively than the other phosphorylation substrates after AMPK activation in the cells. On the other hand, we could not detect the enzymatic activity of the wild-type GFAT1 and the S243A mutant recovered by immunoprecipitation (data not shown), and thus the cell lysates were employed for the measurement of the enzymatic activity. The expression of the S243A mutant attenuated the 2-DG-induced inhibition of GFAT slightly but reproducibly (Fig. 5). The phosphorylation of the endogenous GFAT was not detectable in the cell lysates by the anti-p243 antibody, which recognizes the FLAG-GFAT1 (WT) but does not react with FLAG-GFAT1 (S243A) immunoprecipitated from the transfected cells (Fig. 3B). This phosphorylation site-specific antibody was less sensitive than the PAS antibody as shown in Fig. 3B, and thus it could not detect the endogenous GFAT1 in the cell lysates phosphorylated at Ser243. It is, however, reasonable to assume that the expression of the mutant prevents even in part the phosphorylation of the endogenous GFAT. Presumably, the S243A mutant could interfere in the association of the wild-type GFAT1 with AMPK. The direct evidence is thus unavailable, but the results obtained indicate that the phosphorylation of GFAT1 at Ser243 by AMPK, even partially, reduces the enzymatic activity of GFAT1. It might be possible that AMPK has another indirect influence towards GFAT as the catalytic is still inhibited by 2-DG in the cells expressing the S243A mutant. Further studies are required for the role of AMPK in the regulation of GFAT.
Concerning the regulation by the phosphorylation reaction, GFAT recovered from rat aortic smooth muscle cells treated with cAMP-elevating agents had been shown to have an increase enzymatic activity (Zhou et al. 1998). Later, GFAT1 was confirmed to be phosphorylated by cAMP-dependent protein kinase (PKA) in vitro at Ser205 and Ser235 (Chang et al. 2000), and the former is conserved as Ser202 but the latter does not exist in GFAT2. Mutagenesis studies revealed that the phosphorylation at Ser205 inhibits the enzymatic activity of GFAT1 (Chang et al. 2000), whereas the phosphorylation of GFAT2 by PKA increases its enzymatic activity (Hu et al. 2004). On the other hand, the modification at Ser235 has no obvious effect on the catalytic activity of GFAT1. Therefore, the conflicting results were obtained for the role of the cAMP-mediated regulation of the GFAT isoforms.
On the other hand, the phosphorylation of GFAT1 at Ser243 was first detected as one of 6600 phosphorylation sites on 2244 proteins after stimulating HeLa cells with epidermal growth factor by the dynamic phosphoproteome study (Olsen et al. 2006). Strictly speaking, the phosphorylation at Ser261 of a splicing variant of GFAT1, which corresponds to Ser243 of GFAT1, was found out. Recently, Ser243 was reported as a phosphorylation site in GFAT1 over-expressed in insect cells by mass spectrometric analysis (Li et al. 2007). This group picked out two enzymes, AMPK and Ca2+/calmodulin-dependent protein kinase II (CaMKII), as the candidates for the phosphorylation reaction of Ser243 on the basis of the substrate consensus sequences, and showed the in vitro phosphorylation of GFAT1 at Ser243 by these two kinases using the mutant replacing Ser243 by alanine as a control. In this study, Ser243 was identified as the in vivo phosphorylation site by AMPK induced by glucose depletion in cultured mammalian cells. We have not examined the effects of epidermal growth factor and ligands that increase the intracellular Ca2+ concentration, but it is interesting to know other signaling pathways, if any, that would stimulate the Ser243 phosphorylation of GFAT1. In the case of insect cells (Li et al. 2007), Ser243 seems to be phosphorylated constitutively, whereas the modification of the endogenous protein at this residue was provoked by glucose deprivation in cultured mammalian cells as shown in this study. It is possible to explain this discrepancy, if the insect cells were exposed to some stress that brings AMPK activation. Importantly, Li et al. (2007) proposed that the in vitro phosphorylation at Ser243 results in an increase of the enzymatic activity of GFAT1 based on the comparison of the wild type and the mutant replacing Ser243 by glutamic acid obtained from insect cells, whereas the results in this study indicate that the enzymatic activity of GFAT1 was reduced by the phosphorylation reaction at Ser243. It is reasonable to assume that the enzymatic activity of GFAT1 is lowered by the phosphorylation by AMPK that is activated by glucose depletion, because the amount of UDP-GlcNAc is parallel to glucose availability in the cells. In addition, the activity of GFAT is proposed to be allosterically inhibited by UDP-GlcNAc (Kornfeld 1967; Tourian et al. 1983), the end product of hexosamine signaling pathway, and thus GFAT seems to be regulated by multiple mechanisms. It is thus necessary to clarify the regulation mechanisms of GFAT as well as their relationships under the different conditions to elucidate the biological functions of the hexosamine signaling pathway.
Hyperglycemia-induced insulin resistance, which is defined as the reduced ability of insulin to lower the plasma glucose level, is a hallmark characteristic of type 2 diabetes. The hexosamine signaling pathway has been implicated in glucose-induced insulin resistance (Buse 2006; Copeland et al. 2008). Under hyperglycemia conditions, glucosamine-6-phosphate is formed and rapidly converted to UDP-GlcNAc, and accumulating evidence indicates that abnormal glycosylation with O-GlcNAc is closely correlated to insulin resistance. Of particular interest is that insulin receptor substrate (IRS)-1 and -2, and glucose-transporter type 4 (GLUT4) are glycosylated with O-GlcNAc (Slawson et al. 2006; Zachara & Hart 2006). It is reported that unusual glycosylation with O-GlcNAc of IRS-1 and -2 as well as GLUT4 impairs glucose transport, and is directly linked to the insulin resistance. GFAT is the key molecule in the UDP-GlcNAc synthesis pathway, and thus GFAT could be a potential therapeutic target in the treatment of type 2 diabetes. This study revealed that the hexosamine signaling pathway is regulated by AMPK through the phosphorylation of GFAT1 at Ser243. AMPK is known to operate in coordination with mTOR. Further investigations are required to clarify the relationship among the glycosylation and phosphorylation pathways in the regulation of the cellular activities.
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Experimental procedures |
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The cDNA encoding human GFAT1 (NCBI accession number NM_002056
[GenBank]
) was amplified by PCR with following primers: 5'-GCCGAATTCATGTGTGGTATATTTGCTTAC-3' and 5'-GCCCTCGAGTCACTCTACAGTCACAGATTT-3', where underlined sequences indicate the restriction enzyme sites added, employing Quickclone human brain cDNA library as a template. The product was cloned into pcDNA3 vector with FLAG- or myc-epitope tag at the 5' end, and the resulting vectors were designated as FLAG-GFAT1 (WT), and myc-GFAT1 (WT), respectively. The point mutant of GFAT1 replacing Ser243 by Ala was generated using the QuickchangeTM site-directed mutagenesis kit (Stratagene) using FLAG-GFAT1 (WT) as a template, and the resulting expression vector was designed as FLAG-GFAT1 (S243A). Non-tagged GFAT1 (WT) and its point mutant, GFAT1 (S243A), were generated by PCR with following primers: 5'-CACCGAAT TCGCCACCATGGTGTGGTATATTTGCTTAC-3' and 5'-CCGGAATTCTCACTCTACAGTCACAGATTT-3', where the underlined sequences indicate the restriction enzyme sites added, and the double-underlined sequence indicates a stop codon site, respectively, using FLAG-GFAT1 (WT) and FLAG-GFAT1 (S243A) as templates. The product was cloned into pcDNA3.1 D/V5-His-TOPO vector. The vectors thus generated encode the GFAT1 sequence without His epitope tag by the addition of the stop codon. The pEBG vector of the glutathione S-transferase (GST)-fused constitutively active form of AMPK 1 [GST-AMPK (1-312)] was described previously (Kimura et al. 2003). The pEBG vectors of the GST-fused wild-type AMPK 1 [GST-AMPK (WT)] and kinase-negative form of AMPK 1 [GST-AMPK (K45R)] (Dyck et al. 1996) were kindly provided by Dr Lee A. Witters (Dartmouth Medical School).
Antibodies
The following antibodies were purchased from commercial sources. Normal rabbit immunoglobulin and anti-p70 S6 kinase antibody (H-9) were from Santa Cruz Biotechnology; anti-phospho-Akt substrate (110B7) and anti-phospho-p70 S6 kinase (T389) (1A5) antibodies were from Cell Signaling Technology; anti-phospho-acetyl-CoA carboxylase (S79) and anti-GST antibodies were from Upstate Biotechnology; anti-FLAG (M2) and anti--tubulin antibodies were from Sigma; anti-myc antibody (9E10) was from Roche Applied Science; anti-β-actin antibody was from Abcam. The horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies were from Bio-Rad. The antibodies against GFAT and phosphorylated GFAT1 at Ser243 (anti-pS243) were produced by the antibody service of Immuno-Biological Laboratories against the peptides LRGYDVDFPRNLAKSVTVE (amino acid 663–681) and CNLSRVDpSTT(C)L (amino acids 236–247), where pS and (C) indicate phospho-Ser and aminomethylated-cysteine, respectively. The antibody against phosphorylated GBF1 at Ser243 (pGBF1 (S243)) was produced as described previously (Miyamoto et al. 2008).
Cell culture, transfection and treatment
CHO-IR and HEK293 cells were maintained in Ham's F12 medium (Sigma) and Dulbecco's modified Eagle's medium (DMEM, Sigma), respectively, containing 10% fetal bovine serum (FBS, Invitrogen) at 37 °C in a 5% CO2 incubator. For nutritional starvation, CHO-IR cells were deprived of FBS for 16 h and were further cultured in Dulbecco's phosphate-buffered saline (D-PBS, Invitrogen) for 90 min. Then, the cells were incubated for 30 min with D-PBS or DMEM. For the treatment with 2-DG (Nacalai Tesque), the cells with or without transfection with each expression vector by the lipofection using Lipofectamine reagent (Invitrogen) according to the manufacturer's protocol were cultured in the presence of FBS and replaced in DMEM lacking glucose supplemented with 5.5 mM 2-DG (Nacalai Tesque) for indicated time (Miyamoto et al. 2008). Where indicated, the cells were preincubated with 20 µM Compound C (Merck) in DMEM before the 2-DG treatment.
Protein purification
Immunoprecipitation was carried out at 0–4 °C essentially as described (Hara et al. 1997). Briefly, cells were lysed in buffer A [20 mM Tris-HCl (pH 7.4), 120 mM NaCl, 1 mM EDTA, 5 mM EGTA, 50 mM β-glycerophosphate, 50 mM NaF, 0.3% 3-[(3-cholamidopropyl) dimethylammonio]-1-pro-panesulfonic acid (CHAPS), 1 mM dithiothreitol, 4 µg/mL leupeptin, 4 µg/mL aprotinin], and the lysate was incubated with each antibody and protein G-Sepharose (GE Healthcare Bio-Sciences) for 2 h. As the substrates for the AMPK assay, immunoprecipitated FLAG-GFAT1 (WT) and FLAG-GFAT1 (S243A) from transfected HEK293 cells were eluted by buffer B [60 mM HEPES (pH 7.0), 120 mM NaCl, 2.5 mM β-glycerophosphate, 1 mM dithiothreitol] containing 200 µg/mL of FLAG peptide (Sigma). As the enzymes for the AMPK assay, GST-AMPK (WT) and GST-AMPK (K45R) were expressed in HEK293 cells. After incubation with or without 2-DG for 30 min, the cells were lysed in buffer C [50 mM Tri-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 5 mM sodium diphosphate, 20 mM β-glycerophosphate, 50 mM NaF, 1% Triton X-100, 1 mM DTT, 4 µg/mL leupeptin, 4 µg/mL aprotinin]. After centrifugation, the supernatants were subjected to GST-pull down using glutathione-Sepharose (GE Healthcare Bio-Sciences). The proteins pulled down were washed twice with buffer B, and eluted by buffer B containing 20 mM glutathione.
Protein identification by mass spectrometry
Product ion mass fingerprinting analysis was carried out as described (Oshiro et al. 2007). Briefly, the immunoprecipitates with the PAS antibody from CHO-IR cells were separated by SDS-PAGE, and visualized by silver staining. The protein bands were cut out and subjected to in-gel digestion with trypsin. Resulting peptides were analyzed by the liquid chromatography electrospray ionization mass spectrometry/mass spectrometry (MS/MS) using LCQ Advantage ion trap mass spectrometer (Thermo Finnigan). Protein identification according to product ion mass lists was performed by the product ion mass fingerprinting using MASCOT ‘MS/MS ion search.’
Immunoblot
The lysates, immunoprecipitates, and eluates were separated by SDS-PAGE, and the proteins were transferred on to a polyvinylidene difluoride membrane and subjected to immunoblot using each primary antibody. After incubation with the horseradish peroxidase-conjugated secondary antibody, the proteins were visualized by the enhanced chemiluminescence method. When the same sample was analyzed with different antibodies, the membrane was stripped and employed for the subsequent immunoblot analysis. The results shown are representative of three independent experiments.
AMPK assay
The procedure was carried out essentially as described (Kimura et al. 2003). Purified FLAG-GFAT1 proteins were incubated with either GST-AMPK (WT) or GST-AMPK (K45R) obtained from the 2-DG treated and control HEK293 cells in the AMPK assay mixture [60 mM HEPES (pH 7.0), 120 mM NaCl, 1 mM dithiothreitol, 2.5 mM
β-glycerophosphate, 5 mM MgCl2, 20 µM ATP, 0.2 mM AMP, 5 µCi [-32P]ATP] for 30 min at 30 °C. The reaction was stopped by adding SDS-sample buffer, and the proteins were separated by SDS-PAGE, transferred on to a polyvinylidene difluoride membrane, and analyzed using Bioimaging Analyzer BAS-2500 (Fujifilm). Then the membrane was immunoblotted with appropriate antibodies and visualized as described above.
Enzymatic activity assay of GFAT
The enzymatic activity of GFAT was assayed essentially according to the glutamate dehydrogenase method (Marshall et al. 1991; Ye et al. 2004). Briefly, cells were lysed in buffer A, and the aliquots of the cell lysate were incubated in 100 µL of the reaction mixture (10 mM fructose-6-phosphate, 6 mM glutamine, 0.3 mM APAD, 50 mM KCl, 100 mM KH2PO4, 6 U of glutamate dehydrogenase) at 30 °C for 2 h in a 96-well plate. The change in absorbance by reduction of APAD was monitored at 370 nm by Spectra Max microplate spectrometer (Molecular Devices). The absorbance values of the reaction mixture containing buffer A instead of the cell lysate were employed as reference.
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Acknowledgements |
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Footnotes |
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aPresent address: Department of Pathology and Immunology, Akita University School of Medicine, Akita 010-8543, Japan.
bPresent address: Cancer Medicine and Biophysics Division, National Cancer Center Research Institute, Tokyo 104-0045, Japan.
Deceased on 8 July 2005
* Correspondence: ukikkawa{at}kobe-u.ac.jp |
References |
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Broschat, K.O., Gorka, C., Page, J.D., Martin-Berger, C.L., Davies, M.S., Huang Hc, H.C., Gulve, E.A., Salsgiver, W.J. & Kasten, T.P. (2002) Kinetic characterization of human glutamine–fructose-6-phosphate amidotransferase I: potent feedback inhibition by glucosamine 6-phosphate. J. Biol. Chem. 277, 14764–14770.
Buse, M.G. (2006) Hexosamines, insulin resistance, and the complications of diabetes: current status. Am. J. Physiol. Endocrinol Metab. 290, E1–E8.
Chang, Q., Su, K., Baker, J.R., Yang, X., Paterson, A.J. & Kudlow, J.E. (2000) Phosphorylation of human glutamine : fructose-6-phosphate amidotransferase by cAMP-dependent protein kinase at serine 205 blocks the enzyme activity. J. Biol. Chem. 275, 21981–21987.
Comer, F.I. & Hart, G.W. (2000) O-Glycosylation of nuclear and cytosolic proteins. Dynamic interplay between O-GlcNAc and O-phosphate. J. Biol. Chem. 275, 29179–29182.
Copeland, R.J., Bullen, J.W. & Hart, G.W. (2008) Cross-talk between GlcNAcylation and phosphorylation: roles in insulin resistance and glucose toxicity. Am. J. Physiol. Endocrinol. Metab. 295, E17–E28.
Dyck, J.R., Gao, G., Widmer, J., Stapleton, D., Fernandez, C.S., Kemp, B.E. & Witters, L.A. (1996) Regulation of 5'-AMP-activated protein kinase activity by the noncatalytic β and subunits. J. Biol. Chem. 271, 17798–17803.
Fujita, N., Sato, S., Katayama, K. & Tsuruo, T. (2002) Akt-dependent phosphorylation of p27Kip1 promotes binding to 14–3–3 and cytoplasmic localization. J. Biol. Chem. 277, 28706–28713.
Gwinn, D.M., Shackelford, D.B., Egan, D.F., Mihaylova, M.M., Mery, A., Vasquez, D.S., Turk, B.E. & Shaw, R.J. (2008) AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol. Cell 30, 214–226.[CrossRef][Medline]
Hara, K., Yonezawa, K., Kozlowski, M.T., Sugimoto, T., Andrabi, K., Weng, Q.P., Kasuga, M., Nishimoto, I. & Avruch, J. (1997) Regulation of eIF-4E BP1 phosphorylation by mTOR. J. Biol. Chem. 272, 26457–26463.
Hart, G.W. (1997) Dynamic O-linked glycosylation of nuclear and cytoskeletal proteins. Annu. Rev. Biochem. 66, 315–335.[CrossRef][Medline]
Hu, Y., Riesland, L., Paterson, A.J. & Kudlow, J.E. (2004) Phosphorylation of mouse glutamine-fructose-6-phosphate amidotransferase 2 (GFAT2) by cAMP-dependent protein kinase increases the enzyme activity. J. Biol. Chem. 279, 29988–29993.
Inoki, K., Zhu, T. & Guan, K.L. (2003) TSC2 mediates cellular energy response to control cell growth and survival. Cell 115, 577–590.[CrossRef][Medline]
Kemp, B.E., Stapleton, D., Campbell, D.J., Chen, Z.P., Murthy, S., Walter, M., Gupta, A., Adams, J.J., Katsis, F., van Denderen, B., Jennings, I.G., Iseli, T., Michell, B.J. & Witters, L.A. (2003) AMP-activated protein kinase, super metabolic regulator. Biochem. Soc. Trans. 31, 162–168.[Medline]
Kimura, N., Tokunaga, C., Dalal, S., Richardson, C., Yoshino, K., Hara, K., Kemp, B.E., Witters, L.A., Mimura, O. & Yonezawa, K. (2003) A possible linkage between AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathway. Genes Cells 8, 65–79.[Abstract]
Kornfeld, R. (1967) Studies on L-glutamine D-fructose 6-phosphate amidotransferase. I. Feedback inhibition by uridine diphosphate-N-acetylglucosamine. J. Biol. Chem. 242, 3135–3141.
Kudlow, J.E. (2006) Post-translational modification by O-GlcNAc: another way to change protein function. J. Cell Biochem. 98, 1062–1075.[CrossRef][Medline]
Li, Y., Roux, C., Lazereg, S., LeCaer, J.P., Laprevote, O., Badet, B. & Badet-Denisot, M.A. (2007) Identification of a novel serine phosphorylation site in human glutamine : fructose-6-phosphate amidotransferase isoform 1. Biochemistry 46, 13163–13169.[CrossRef][Medline]
Marshall, S. (2006) Role of insulin, adipocyte hormones, and nutrient-sensing pathways in regulating fuel metabolism and energy homeostasis: a nutritional perspective of diabetes, obesity, and cancer. Sci. STKE 2006, re7.
Marshall, S., Bacote, V. & Traxinger, R.R. (1991) Discovery of a metabolic pathway mediating glucose-induced desensitization of the glucose transport system. Role of hexosamine biosynthesis in the induction of insulin resistance. J. Biol. Chem. 266, 4706–4712.
Massiere, F. & Badet-Denisot, M.A. (1998) The mechanism of glutamine-dependent amidotransferases. Cell Mol. Life Sci. 54, 205–222.[CrossRef][Medline]
McKnight, G.L., Mudri, S.L., Mathewes, S.L., Traxinger, R.R., Marshall, S., Sheppard, P.O. & O’Hara, P.J. (1992) Molecular cloning, cDNA sequence, and bacterial expression of human glutamine : fructose-6-phosphate amidotransferase. J. Biol. Chem. 267, 25208–25212.
Miyamoto, T., Oshiro, N., Yoshino, K., Nakashima, A., Eguchi, S., Takahashi, M., Ono, Y., Kikkawa, U. & Yonezawa, K. (2008) AMP-activated protein kinase phosphorylates Golgi-specific brefeldin A resistance factor 1 at Thr1337 to induce disassembly of Golgi apparatus. J. Biol. Chem. 283, 4430–4438.
Oki, T., Yamazaki, K., Kuromitsu, J., Okada, M. & Tanaka, I. (1999) cDNA cloning and mapping of a novel subtype of glutamine : fructose-6-phosphate amidotransferase (GFAT2) in human and mouse. Genomics 57, 227–234.[CrossRef][Medline]
Olsen, J.V., Blagoev, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P. & Mann, M. (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635–648.[CrossRef][Medline]
Oshiro, N., Takahashi, R., Yoshino, K., Tanimura, K., Nakashima, A., Eguchi, S., Miyamoto, T., Hara, K., Takehana, K., Avruch, J., Kikkawa, U. & Yonezawa, K. (2007) The proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target of rapamycin complex 1. J. Biol. Chem. 282, 20329–20339.
Slawson, C. & Hart, G.W. (2003) Dynamic interplay between O-GlcNAc and O-phosphate: the sweet side of protein regulation. Curr. Opin. Struct. Biol. 13, 631–636.[CrossRef][Medline]
Slawson, C., Housley, M.P. & Hart, G.W. (2006) O-GlcNAc cycling: how a single sugar post-translational modification is changing the way we think about signaling networks. J. Cell Biochem. 97, 71–83.[CrossRef][Medline]
Teplyakov, A., Obmolova, G., Badet, B. & Badet-Denisot, M.A. (2001) Channeling of ammonia in glucosamine-6-phosphate synthase. J. Mol. Biol. 313, 1093–1102.[CrossRef][Medline]
Tourian, A., Callahan, M. & Hung, W. (1983) L-glutamine D-fructose-6-P aminotransferase regulation by glucose-6-P and UDP-N-acetylglucosamine. Neurochem. Res. 8, 1589–1595.[CrossRef][Medline]
Towler, M.C. & Hardie, D.G. (2007) AMP-activated protein kinase in metabolic control and insulin signaling. Circ. Res. 100, 328–341.
Yang, Q. & Guan, K.L. (2007) Expanding mTOR signaling. Cell Res. 17, 666–681.[CrossRef][Medline]
Ye, F., Maegawa, H., Morino, K., Kashiwagi, A., Kikkawa, R., Xie, M. & Shen, Z. (2004) A simple and sensitive method for glutamine : fructose-6-phosphate amidotransferase assay. J. Biochem. Biophys. Methods 59, 201–208.[Medline]
Zachara, N.E. & Hart, G.W. (2006) Cell signaling, the essential role of O-GlcNAc! Biochim. Biophys. Acta 1761, 599–617.
Zhou, G., Myers, R., Li, Y., Chen, Y., Shen, X., Fenyk-Melody, J., Wu, M., Ventre, J., Doebber, T., Fujii, N., Musi, N., Hirshman, M.F., Goodyear, L.J. & Moller, D.E. (2001) Role of AMP-activated protein kinase in mechanism of metformin action. J. Clin. Invest. 108, 1167–1174.[CrossRef][Medline]
Zhou, J., Huynh, Q.K., Hoffman, R.T., Crook, E.D., Daniels, M.C., Gulve, E.A. & McClain, D.A. (1998) Regulation of glutamine : fructose-6-phosphate amidotransferase by cAMP-dependent protein kinase. Diabetes 47, 1836–1840.[Abstract]
Received: 22 September 2008
Accepted: 5 November 2008
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