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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 acetylation is critical for binding and retaining GK in the nucleus.(A,B) Interaction between GKRP and human glucokinase (hGK). HeLa cells transfected with expression vectors for GKRP and hGK were lysed, protein immunoprecipitated with anti-Myc or anti-V5 antibodies, and immunoblotted by anti-His or anti-Myc antibodies. (C) Effect of K5 mutations on GKRP interaction with hGK. HeLa cells cotransfected with hGK and wild type or the indicated GKRP mutants were lysed, protein precipitated with an anti-Myc antibody, and blotted with an anti-His antibody. β-actin expression was used as internal control. (D) Immunofluorescence micrographs showing the subcellular locations of GKRP and hGK. HeLa cells transfected with Myc-tagged GKRP and V5/His-tagged GK were maintained in 5.5 mM or 25 mM glucose for 4 hr in the absence (−) or presence (+) of a mixture of the HDACIs NAM (5 mM) and TSA (1 μM). Data are expressed as means ± SEMs, n = 4, ***p ≤ 0.001.
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f3: GKRP acetylation is critical for binding and retaining GK in the nucleus.(A,B) Interaction between GKRP and human glucokinase (hGK). HeLa cells transfected with expression vectors for GKRP and hGK were lysed, protein immunoprecipitated with anti-Myc or anti-V5 antibodies, and immunoblotted by anti-His or anti-Myc antibodies. (C) Effect of K5 mutations on GKRP interaction with hGK. HeLa cells cotransfected with hGK and wild type or the indicated GKRP mutants were lysed, protein precipitated with an anti-Myc antibody, and blotted with an anti-His antibody. β-actin expression was used as internal control. (D) Immunofluorescence micrographs showing the subcellular locations of GKRP and hGK. HeLa cells transfected with Myc-tagged GKRP and V5/His-tagged GK were maintained in 5.5 mM or 25 mM glucose for 4 hr in the absence (−) or presence (+) of a mixture of the HDACIs NAM (5 mM) and TSA (1 μM). Data are expressed as means ± SEMs, n = 4, ***p ≤ 0.001.

Mentions: Next, we performed ubiquitin degradation assays using each of the acetyl- mimic and deacetyl-mimic forms of GKRP. As shown in Fig. 3A,B, GK robustly interacted with the WT and the K5Q GKRP acetyl-mimic, but significantly less with the K5R GKRP deacetyl-mimic (Fig. 3C,D, p ≤ 0.001). Moreover, acetylated GKRP showed increased interaction with GK in vitro (Supplementary Fig. 3A,B). From these results, we speculated that GKRP acetylation promotes its interaction with GK. As a result, GK-GKRP complex formation is believed to be critical for regulating GK activity and cytosolic glycolysis, consistent with a previous finding that GKRP acetylation caused GK nuclear retention22. To visualize whether GKRP K5 acetylation affects nuclear retention of the GKRP-GK complex, due to glucose “master sensors”, HeLa cells were incubated in 5.5 mM glucose, and immunofluorescence microscopy then performed (Fig. 3E, upper panel). As shown, most of the GK and GKRP localized to the nucleus. HeLa cells incubation in 25 mM glucose, however, resulted in the presence of both GK and GKRP in the cytosol (Fig. 3E, middle panel), consistent with other studies of this phenomenon23. As HDACI treatment similarly increased nuclear retention of the complex (Fig. 3E, lower panel), taken together, these results solidly suggest that GKRP acetylation increases nuclear retention of GK.


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 acetylation is critical for binding and retaining GK in the nucleus.(A,B) Interaction between GKRP and human glucokinase (hGK). HeLa cells transfected with expression vectors for GKRP and hGK were lysed, protein immunoprecipitated with anti-Myc or anti-V5 antibodies, and immunoblotted by anti-His or anti-Myc antibodies. (C) Effect of K5 mutations on GKRP interaction with hGK. HeLa cells cotransfected with hGK and wild type or the indicated GKRP mutants were lysed, protein precipitated with an anti-Myc antibody, and blotted with an anti-His antibody. β-actin expression was used as internal control. (D) Immunofluorescence micrographs showing the subcellular locations of GKRP and hGK. HeLa cells transfected with Myc-tagged GKRP and V5/His-tagged GK were maintained in 5.5 mM or 25 mM glucose for 4 hr in the absence (−) or presence (+) of a mixture of the HDACIs NAM (5 mM) and TSA (1 μM). Data are expressed as means ± SEMs, n = 4, ***p ≤ 0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664969&req=5

f3: GKRP acetylation is critical for binding and retaining GK in the nucleus.(A,B) Interaction between GKRP and human glucokinase (hGK). HeLa cells transfected with expression vectors for GKRP and hGK were lysed, protein immunoprecipitated with anti-Myc or anti-V5 antibodies, and immunoblotted by anti-His or anti-Myc antibodies. (C) Effect of K5 mutations on GKRP interaction with hGK. HeLa cells cotransfected with hGK and wild type or the indicated GKRP mutants were lysed, protein precipitated with an anti-Myc antibody, and blotted with an anti-His antibody. β-actin expression was used as internal control. (D) Immunofluorescence micrographs showing the subcellular locations of GKRP and hGK. HeLa cells transfected with Myc-tagged GKRP and V5/His-tagged GK were maintained in 5.5 mM or 25 mM glucose for 4 hr in the absence (−) or presence (+) of a mixture of the HDACIs NAM (5 mM) and TSA (1 μM). Data are expressed as means ± SEMs, n = 4, ***p ≤ 0.001.
Mentions: Next, we performed ubiquitin degradation assays using each of the acetyl- mimic and deacetyl-mimic forms of GKRP. As shown in Fig. 3A,B, GK robustly interacted with the WT and the K5Q GKRP acetyl-mimic, but significantly less with the K5R GKRP deacetyl-mimic (Fig. 3C,D, p ≤ 0.001). Moreover, acetylated GKRP showed increased interaction with GK in vitro (Supplementary Fig. 3A,B). From these results, we speculated that GKRP acetylation promotes its interaction with GK. As a result, GK-GKRP complex formation is believed to be critical for regulating GK activity and cytosolic glycolysis, consistent with a previous finding that GKRP acetylation caused GK nuclear retention22. To visualize whether GKRP K5 acetylation affects nuclear retention of the GKRP-GK complex, due to glucose “master sensors”, HeLa cells were incubated in 5.5 mM glucose, and immunofluorescence microscopy then performed (Fig. 3E, upper panel). As shown, most of the GK and GKRP localized to the nucleus. HeLa cells incubation in 25 mM glucose, however, resulted in the presence of both GK and GKRP in the cytosol (Fig. 3E, middle panel), consistent with other studies of this phenomenon23. As HDACI treatment similarly increased nuclear retention of the complex (Fig. 3E, lower panel), taken together, these results solidly suggest that GKRP acetylation increases nuclear retention of GK.

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