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Acetylation of glucokinase regulatory protein decreases glucose metabolism by suppressing glucokinase activity.

Park JM, Kim TH, Jo SH, Kim MY, Ahn YH - Sci Rep (2015)

Bottom Line: Post-translationally, GK is regulated by binding the glucokinase regulatory protein (GKRP), resulting in GK retention in the nucleus and its inability to participate in cytosolic glycolysis.Acetylated GKRP is resistant to degradation by the ubiquitin-dependent proteasome pathway, suggesting that acetylation increases GKRP stability and binding to GK, further inhibiting GK nuclear export.Deacetylation of GKRP is effected by the NAD(+)-dependent, class III histone deacetylase SIRT2, which is inhibited by nicotinamide.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Republic of Korea.

ABSTRACT
Glucokinase (GK), mainly expressed in the liver and pancreatic β-cells, is critical for maintaining glucose homeostasis. GK expression and kinase activity, respectively, are both modulated at the transcriptional and post-translational levels. Post-translationally, GK is regulated by binding the glucokinase regulatory protein (GKRP), resulting in GK retention in the nucleus and its inability to participate in cytosolic glycolysis. Although hepatic GKRP is known to be regulated by allosteric mechanisms, the precise details of modulation of GKRP activity, by post-translational modification, are not well known. Here, we demonstrate that GKRP is acetylated at Lys5 by the acetyltransferase p300. Acetylated GKRP is resistant to degradation by the ubiquitin-dependent proteasome pathway, suggesting that acetylation increases GKRP stability and binding to GK, further inhibiting GK nuclear export. Deacetylation of GKRP is effected by the NAD(+)-dependent, class III histone deacetylase SIRT2, which is inhibited by nicotinamide. Moreover, the livers of db/db obese, diabetic mice also show elevated GKRP acetylation, suggesting a broader, critical role in regulating blood glucose. Given that acetylated GKRP may affiliate with type-2 diabetes mellitus (T2DM), understanding the mechanism of GKRP acetylation in the liver could reveal novel targets within the GK-GKRP pathway, for treating T2DM and other metabolic pathologies.

No MeSH data available.


Related in: MedlinePlus

GKRP is acetylated by p300.(A) Effects of histone deacetylase inhibitors on GKRP. HeLa cells transfected with Myc-tagged GKRP were treated with 5 mM NAM and 1 μM TSA 6 hr before harvest. (B) Band intensities of acetylated Myc-GKRP were quantified by Image J software. The values from samples not treated with NAM and TSA were set to 1.0. Data are shown as the means ± SEM of four independent experiments. (C) Identification of the acetyltransferase responsible for GKRP acetylation. Expression vectors of various acetyltransferases (ATs) were co-transfected with pSG-Myc GKRP into HeLa cells. The immunoprecipitates (from antibodies against the various ATs) were then subjected to immunoblot with antibodies against Ac-Lys or Myc. (D) LC-MS/MS spectrum of GKRP peptides showing that acetylation occurs at K5. (E) Effects of site-specific mutation on the potential acetylation site, GKRP K5. Substitutions of Lys (K) with Arg (R) or Glu (Q) at the indicated sites are shown in parenthesis. HeLa cells transfected with the indicated mutant or wild-type plasmids were lysed and immunoprecipitated by an anti-Myc antibody. Acetylated GKRP was detected by an anti-Ac-Lys antibody. (F) Sequence alignment of the GKRP region containing K5 from various species. NAM, nicotinamide; TSA, Trichostatin A. Data are expressed as means ± SEMs, n = 4, *p ≤ 0.05; **p ≤ 0.01.
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f1: GKRP is acetylated by p300.(A) Effects of histone deacetylase inhibitors on GKRP. HeLa cells transfected with Myc-tagged GKRP were treated with 5 mM NAM and 1 μM TSA 6 hr before harvest. (B) Band intensities of acetylated Myc-GKRP were quantified by Image J software. The values from samples not treated with NAM and TSA were set to 1.0. Data are shown as the means ± SEM of four independent experiments. (C) Identification of the acetyltransferase responsible for GKRP acetylation. Expression vectors of various acetyltransferases (ATs) were co-transfected with pSG-Myc GKRP into HeLa cells. The immunoprecipitates (from antibodies against the various ATs) were then subjected to immunoblot with antibodies against Ac-Lys or Myc. (D) LC-MS/MS spectrum of GKRP peptides showing that acetylation occurs at K5. (E) Effects of site-specific mutation on the potential acetylation site, GKRP K5. Substitutions of Lys (K) with Arg (R) or Glu (Q) at the indicated sites are shown in parenthesis. HeLa cells transfected with the indicated mutant or wild-type plasmids were lysed and immunoprecipitated by an anti-Myc antibody. Acetylated GKRP was detected by an anti-Ac-Lys antibody. (F) Sequence alignment of the GKRP region containing K5 from various species. NAM, nicotinamide; TSA, Trichostatin A. Data are expressed as means ± SEMs, n = 4, *p ≤ 0.05; **p ≤ 0.01.

Mentions: Since most metabolic enzymes are acetylated18, we assessed GKRP for possible acetylation that might modulate GK activity, in HeLa cells, which do not express GKRP, transfected with a Myc-GKRP fusion expression vector. Treatment with the histone deacetylase inhibitors (HDACIs) nicotinamide (NAM) and Trichostatin A (TSA)19 notably increased GKRP acetylation (Fig. 1A,B, p ≤ 0.05). To identify the acetyltransferase(s) responsible for GKRP acetylation, HeLa cells were cotransfected with expression vectors for Myc-GKRP and various acetyltransferases, including the General CoNtrol of amino synthesis (GCN5, KAT2), p300/CBP-associated factor (PCAF, KAT2B), HIV-1 Tat interactive protein 60 kDa (Tip60), human MYST histone acetyltransferase 1 (hMOF, KAT8), CREB-binding protein (CBP, CREBB2), or p300 (EP300). As shown in Fig. 1C, GKRP was predominantly acetylated by p300, followed by hMOF, in a dose-dependent manner (Supplementary Fig. S2A). Moreover, p300 and GKRP directly interacted with each other, as shown by co-immunoprecipitation (Supplementary Fig. S2B). To test whether p300 plays a role in regulating GKRP acetylation, we used C646, a p300-specific inhibitor20, to treat HeLa cells transfected with expression vectors for Myc-GKRP and Flag-p300. That assessment demonstrated that C646 treatment decreased GKRP acetylation (Supplementary Fig. S2C), indicating that p300 acetylates GKRP.


Acetylation of glucokinase regulatory protein decreases glucose metabolism by suppressing glucokinase activity.

Park JM, Kim TH, Jo SH, Kim MY, Ahn YH - Sci Rep (2015)

GKRP is acetylated by p300.(A) Effects of histone deacetylase inhibitors on GKRP. HeLa cells transfected with Myc-tagged GKRP were treated with 5 mM NAM and 1 μM TSA 6 hr before harvest. (B) Band intensities of acetylated Myc-GKRP were quantified by Image J software. The values from samples not treated with NAM and TSA were set to 1.0. Data are shown as the means ± SEM of four independent experiments. (C) Identification of the acetyltransferase responsible for GKRP acetylation. Expression vectors of various acetyltransferases (ATs) were co-transfected with pSG-Myc GKRP into HeLa cells. The immunoprecipitates (from antibodies against the various ATs) were then subjected to immunoblot with antibodies against Ac-Lys or Myc. (D) LC-MS/MS spectrum of GKRP peptides showing that acetylation occurs at K5. (E) Effects of site-specific mutation on the potential acetylation site, GKRP K5. Substitutions of Lys (K) with Arg (R) or Glu (Q) at the indicated sites are shown in parenthesis. HeLa cells transfected with the indicated mutant or wild-type plasmids were lysed and immunoprecipitated by an anti-Myc antibody. Acetylated GKRP was detected by an anti-Ac-Lys antibody. (F) Sequence alignment of the GKRP region containing K5 from various species. NAM, nicotinamide; TSA, Trichostatin A. Data are expressed as means ± SEMs, n = 4, *p ≤ 0.05; **p ≤ 0.01.
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Related In: Results  -  Collection

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Show All Figures
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f1: GKRP is acetylated by p300.(A) Effects of histone deacetylase inhibitors on GKRP. HeLa cells transfected with Myc-tagged GKRP were treated with 5 mM NAM and 1 μM TSA 6 hr before harvest. (B) Band intensities of acetylated Myc-GKRP were quantified by Image J software. The values from samples not treated with NAM and TSA were set to 1.0. Data are shown as the means ± SEM of four independent experiments. (C) Identification of the acetyltransferase responsible for GKRP acetylation. Expression vectors of various acetyltransferases (ATs) were co-transfected with pSG-Myc GKRP into HeLa cells. The immunoprecipitates (from antibodies against the various ATs) were then subjected to immunoblot with antibodies against Ac-Lys or Myc. (D) LC-MS/MS spectrum of GKRP peptides showing that acetylation occurs at K5. (E) Effects of site-specific mutation on the potential acetylation site, GKRP K5. Substitutions of Lys (K) with Arg (R) or Glu (Q) at the indicated sites are shown in parenthesis. HeLa cells transfected with the indicated mutant or wild-type plasmids were lysed and immunoprecipitated by an anti-Myc antibody. Acetylated GKRP was detected by an anti-Ac-Lys antibody. (F) Sequence alignment of the GKRP region containing K5 from various species. NAM, nicotinamide; TSA, Trichostatin A. Data are expressed as means ± SEMs, n = 4, *p ≤ 0.05; **p ≤ 0.01.
Mentions: Since most metabolic enzymes are acetylated18, we assessed GKRP for possible acetylation that might modulate GK activity, in HeLa cells, which do not express GKRP, transfected with a Myc-GKRP fusion expression vector. Treatment with the histone deacetylase inhibitors (HDACIs) nicotinamide (NAM) and Trichostatin A (TSA)19 notably increased GKRP acetylation (Fig. 1A,B, p ≤ 0.05). To identify the acetyltransferase(s) responsible for GKRP acetylation, HeLa cells were cotransfected with expression vectors for Myc-GKRP and various acetyltransferases, including the General CoNtrol of amino synthesis (GCN5, KAT2), p300/CBP-associated factor (PCAF, KAT2B), HIV-1 Tat interactive protein 60 kDa (Tip60), human MYST histone acetyltransferase 1 (hMOF, KAT8), CREB-binding protein (CBP, CREBB2), or p300 (EP300). As shown in Fig. 1C, GKRP was predominantly acetylated by p300, followed by hMOF, in a dose-dependent manner (Supplementary Fig. S2A). Moreover, p300 and GKRP directly interacted with each other, as shown by co-immunoprecipitation (Supplementary Fig. S2B). To test whether p300 plays a role in regulating GKRP acetylation, we used C646, a p300-specific inhibitor20, to treat HeLa cells transfected with expression vectors for Myc-GKRP and Flag-p300. That assessment demonstrated that C646 treatment decreased GKRP acetylation (Supplementary Fig. S2C), indicating that p300 acetylates GKRP.

Bottom Line: Post-translationally, GK is regulated by binding the glucokinase regulatory protein (GKRP), resulting in GK retention in the nucleus and its inability to participate in cytosolic glycolysis.Acetylated GKRP is resistant to degradation by the ubiquitin-dependent proteasome pathway, suggesting that acetylation increases GKRP stability and binding to GK, further inhibiting GK nuclear export.Deacetylation of GKRP is effected by the NAD(+)-dependent, class III histone deacetylase SIRT2, which is inhibited by nicotinamide.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Republic of Korea.

ABSTRACT
Glucokinase (GK), mainly expressed in the liver and pancreatic β-cells, is critical for maintaining glucose homeostasis. GK expression and kinase activity, respectively, are both modulated at the transcriptional and post-translational levels. Post-translationally, GK is regulated by binding the glucokinase regulatory protein (GKRP), resulting in GK retention in the nucleus and its inability to participate in cytosolic glycolysis. Although hepatic GKRP is known to be regulated by allosteric mechanisms, the precise details of modulation of GKRP activity, by post-translational modification, are not well known. Here, we demonstrate that GKRP is acetylated at Lys5 by the acetyltransferase p300. Acetylated GKRP is resistant to degradation by the ubiquitin-dependent proteasome pathway, suggesting that acetylation increases GKRP stability and binding to GK, further inhibiting GK nuclear export. Deacetylation of GKRP is effected by the NAD(+)-dependent, class III histone deacetylase SIRT2, which is inhibited by nicotinamide. Moreover, the livers of db/db obese, diabetic mice also show elevated GKRP acetylation, suggesting a broader, critical role in regulating blood glucose. Given that acetylated GKRP may affiliate with type-2 diabetes mellitus (T2DM), understanding the mechanism of GKRP acetylation in the liver could reveal novel targets within the GK-GKRP pathway, for treating T2DM and other metabolic pathologies.

No MeSH data available.


Related in: MedlinePlus