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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

Ubiquitin-dependent GKRP degradation is decreased by acetylation.(A) Effects of histone deacetylase inhibitors on GKRP levels in. HeLa cells transfected with Myc-tagged GKRP. (B) Effect of p300 on GKRP protein levels in HeLa cells transfected with Myc-tagged GKRP and full-length p300. (C) Effect of anti-p300 siRNA on GKRP protein levels. HeLa cells transfected with Myc-tagged GKRP and negative control or p300 siRNA (10 nM) were incubated for 48 hr, and GKRP protein levels assessed by immunoblot using an anti-Myc antibody. (D) Effect of ubiquitin on GKRP stability. HeLa cells cotransfected with Myc-tagged GKRP and HA-tagged ubiquitin were maintained in the absence or presence of MG132 (10 μM) for 2 hr. Cell lysates were precipitated with an anti-Myc antibody and immunoblotting used to detect ubquitinated GKRP by anti-HA antibody. (E) Effect of various K5 mutants on GKRP ubiquitination. Myc-tagged wild type, deacetyl-mimic (K5R), or acetyl-mimic (K5Q) GKRP was cotransfected with HA-ubiquitin in the presence of the proteasome inhibitor MG132 (10 μM) for 2 hr. Cell lysates were precipitated by an anti-Myc antibody, and immunoblot with an anti-HA antibody used to detect ubiquitinated GKRP. (F) Effects of HDACIs on GKRP stability. HeLa cells were transfected with Myc-tagged GKRP were treated with NAM (5 mM) and TSA (1 μM) 4 hr before treating CHX. 24 hr after transfection, cells were treated with 50 μg/ml of the protein synthesis inhibitor CHX for the indicated time periods. GKRP protein levels from cells collected at time zero were defined as 100%. GAPDH and β-actin were used as an internal control. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. HDACIs, histone deacetylase inhibitor, NAM, nicotinamide, TSA, Trichostatin A, and CHX, cycloheximide. Data are expressed as means ± SEMs, n = 4, ***p ≤ 0.001.
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f2: Ubiquitin-dependent GKRP degradation is decreased by acetylation.(A) Effects of histone deacetylase inhibitors on GKRP levels in. HeLa cells transfected with Myc-tagged GKRP. (B) Effect of p300 on GKRP protein levels in HeLa cells transfected with Myc-tagged GKRP and full-length p300. (C) Effect of anti-p300 siRNA on GKRP protein levels. HeLa cells transfected with Myc-tagged GKRP and negative control or p300 siRNA (10 nM) were incubated for 48 hr, and GKRP protein levels assessed by immunoblot using an anti-Myc antibody. (D) Effect of ubiquitin on GKRP stability. HeLa cells cotransfected with Myc-tagged GKRP and HA-tagged ubiquitin were maintained in the absence or presence of MG132 (10 μM) for 2 hr. Cell lysates were precipitated with an anti-Myc antibody and immunoblotting used to detect ubquitinated GKRP by anti-HA antibody. (E) Effect of various K5 mutants on GKRP ubiquitination. Myc-tagged wild type, deacetyl-mimic (K5R), or acetyl-mimic (K5Q) GKRP was cotransfected with HA-ubiquitin in the presence of the proteasome inhibitor MG132 (10 μM) for 2 hr. Cell lysates were precipitated by an anti-Myc antibody, and immunoblot with an anti-HA antibody used to detect ubiquitinated GKRP. (F) Effects of HDACIs on GKRP stability. HeLa cells were transfected with Myc-tagged GKRP were treated with NAM (5 mM) and TSA (1 μM) 4 hr before treating CHX. 24 hr after transfection, cells were treated with 50 μg/ml of the protein synthesis inhibitor CHX for the indicated time periods. GKRP protein levels from cells collected at time zero were defined as 100%. GAPDH and β-actin were used as an internal control. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. HDACIs, histone deacetylase inhibitor, NAM, nicotinamide, TSA, Trichostatin A, and CHX, cycloheximide. Data are expressed as means ± SEMs, n = 4, ***p ≤ 0.001.

Mentions: Acetylated GKRP acetylation resists ubiquitin-mediated degradation. To assess the effect of acetylation on GKRP stability, we treated HeLa cells with the aforementioned HDACIs, NAM and TSA, resulting in increased acetylated GKRP protein levels (Fig. 2A). Conversely, immunoblotting demonstrated that Myc-GKRP coexpression with HA-tagged full-length p300 increased, and anti-p300 siRNA decreased, GKRP protein levels (Fig. 2B,C). These results strongly suggest that GKRP acetylation by p300 might mediate its stability.


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)

Ubiquitin-dependent GKRP degradation is decreased by acetylation.(A) Effects of histone deacetylase inhibitors on GKRP levels in. HeLa cells transfected with Myc-tagged GKRP. (B) Effect of p300 on GKRP protein levels in HeLa cells transfected with Myc-tagged GKRP and full-length p300. (C) Effect of anti-p300 siRNA on GKRP protein levels. HeLa cells transfected with Myc-tagged GKRP and negative control or p300 siRNA (10 nM) were incubated for 48 hr, and GKRP protein levels assessed by immunoblot using an anti-Myc antibody. (D) Effect of ubiquitin on GKRP stability. HeLa cells cotransfected with Myc-tagged GKRP and HA-tagged ubiquitin were maintained in the absence or presence of MG132 (10 μM) for 2 hr. Cell lysates were precipitated with an anti-Myc antibody and immunoblotting used to detect ubquitinated GKRP by anti-HA antibody. (E) Effect of various K5 mutants on GKRP ubiquitination. Myc-tagged wild type, deacetyl-mimic (K5R), or acetyl-mimic (K5Q) GKRP was cotransfected with HA-ubiquitin in the presence of the proteasome inhibitor MG132 (10 μM) for 2 hr. Cell lysates were precipitated by an anti-Myc antibody, and immunoblot with an anti-HA antibody used to detect ubiquitinated GKRP. (F) Effects of HDACIs on GKRP stability. HeLa cells were transfected with Myc-tagged GKRP were treated with NAM (5 mM) and TSA (1 μM) 4 hr before treating CHX. 24 hr after transfection, cells were treated with 50 μg/ml of the protein synthesis inhibitor CHX for the indicated time periods. GKRP protein levels from cells collected at time zero were defined as 100%. GAPDH and β-actin were used as an internal control. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. HDACIs, histone deacetylase inhibitor, NAM, nicotinamide, TSA, Trichostatin A, and CHX, cycloheximide. Data are expressed as means ± SEMs, n = 4, ***p ≤ 0.001.
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f2: Ubiquitin-dependent GKRP degradation is decreased by acetylation.(A) Effects of histone deacetylase inhibitors on GKRP levels in. HeLa cells transfected with Myc-tagged GKRP. (B) Effect of p300 on GKRP protein levels in HeLa cells transfected with Myc-tagged GKRP and full-length p300. (C) Effect of anti-p300 siRNA on GKRP protein levels. HeLa cells transfected with Myc-tagged GKRP and negative control or p300 siRNA (10 nM) were incubated for 48 hr, and GKRP protein levels assessed by immunoblot using an anti-Myc antibody. (D) Effect of ubiquitin on GKRP stability. HeLa cells cotransfected with Myc-tagged GKRP and HA-tagged ubiquitin were maintained in the absence or presence of MG132 (10 μM) for 2 hr. Cell lysates were precipitated with an anti-Myc antibody and immunoblotting used to detect ubquitinated GKRP by anti-HA antibody. (E) Effect of various K5 mutants on GKRP ubiquitination. Myc-tagged wild type, deacetyl-mimic (K5R), or acetyl-mimic (K5Q) GKRP was cotransfected with HA-ubiquitin in the presence of the proteasome inhibitor MG132 (10 μM) for 2 hr. Cell lysates were precipitated by an anti-Myc antibody, and immunoblot with an anti-HA antibody used to detect ubiquitinated GKRP. (F) Effects of HDACIs on GKRP stability. HeLa cells were transfected with Myc-tagged GKRP were treated with NAM (5 mM) and TSA (1 μM) 4 hr before treating CHX. 24 hr after transfection, cells were treated with 50 μg/ml of the protein synthesis inhibitor CHX for the indicated time periods. GKRP protein levels from cells collected at time zero were defined as 100%. GAPDH and β-actin were used as an internal control. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. HDACIs, histone deacetylase inhibitor, NAM, nicotinamide, TSA, Trichostatin A, and CHX, cycloheximide. Data are expressed as means ± SEMs, n = 4, ***p ≤ 0.001.
Mentions: Acetylated GKRP acetylation resists ubiquitin-mediated degradation. To assess the effect of acetylation on GKRP stability, we treated HeLa cells with the aforementioned HDACIs, NAM and TSA, resulting in increased acetylated GKRP protein levels (Fig. 2A). Conversely, immunoblotting demonstrated that Myc-GKRP coexpression with HA-tagged full-length p300 increased, and anti-p300 siRNA decreased, GKRP protein levels (Fig. 2B,C). These results strongly suggest that GKRP acetylation by p300 might mediate its stability.

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