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The GCN5-CITED2-PKA signalling module controls hepatic glucose metabolism through a cAMP-induced substrate switch

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ABSTRACT

Hepatic gluconeogenesis during fasting results from gluconeogenic gene activation via the glucagon–cAMP–protein kinase A (PKA) pathway, a process whose dysregulation underlies fasting hyperglycemia in diabetes. Such transcriptional activation requires epigenetic changes at promoters by mechanisms that have remained unclear. Here we show that GCN5 functions both as a histone acetyltransferase (HAT) to activate fasting gluconeogenesis and as an acetyltransferase for the transcriptional co-activator PGC-1α to inhibit gluconeogenesis in the fed state. During fasting, PKA phosphorylates GCN5 in a manner dependent on the transcriptional coregulator CITED2, thereby increasing its acetyltransferase activity for histone and attenuating that for PGC-1α. This substrate switch concomitantly promotes both epigenetic changes associated with transcriptional activation and PGC-1α–mediated coactivation, thereby triggering gluconeogenesis. The GCN5-CITED2-PKA signalling module and associated GCN5 substrate switch thus serve as a key driver of gluconeogenesis. Disruption of this module ameliorates hyperglycemia in obese diabetic animals, offering a potential therapeutic strategy for such conditions.

No MeSH data available.


GCN5 is phosphorylated by PKA within a GCN5-CITED2-PKA signalling module.(a) Effects of PKA inhibition with H89 (20 μM, 6 h) on gluconeogenic gene expression induced by overexpression of PGC-1α with or without CITED2 in primary hepatocytes. Data are means±s.e.m. (n=3). **P<0.01 (ANOVA with Bonferroni's post hoc test). (b) Immunoblot analysis of the effects of HA-CITED2 expression or pCPT-cAMP treatment (10 or 30 min) on phosphorylation of FLAG-GCN5 in AML12 cells as assessed with antibodies to phosphorylated PKA substrates. (c) Effects of CITED2 depletion on pCPT-cAMP-induced phosphorylation of FLAG-GCN5 and other PKA substrates as well as on the dephosphorylation of CRTC2 in primary hepatocytes. (d,e) IP and immunoblot analysis of the interaction of FLAG-GCN5 with Myc-PKAC and HA-CITED2 (d) as well as of the effect of CITED2 knockdown on the interaction of FLAG-GCN5 with PKAC (e) in AML12 cells. (f) Effect of siRNA-mediated PKAC depletion in AML12 cells on basal and CITED2-induced HAT activity of FLAG-GCN5 as assessed by in vitro assay. (g) Effect of PKAC overexpression in AML12 cells on HAT activity of FLAG-GCN5 measured in vitro. A Myc-PKAC plasmid and PKAC siRNA were introduced into cells by transfection, whereas adenoviral vectors were used to introduce other exogenous proteins or shRNAs in these experiments. Data are representative of at least three independent experiments. ANOVA, analysis of variance; siRNA; small interfering RNA.
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f6: GCN5 is phosphorylated by PKA within a GCN5-CITED2-PKA signalling module.(a) Effects of PKA inhibition with H89 (20 μM, 6 h) on gluconeogenic gene expression induced by overexpression of PGC-1α with or without CITED2 in primary hepatocytes. Data are means±s.e.m. (n=3). **P<0.01 (ANOVA with Bonferroni's post hoc test). (b) Immunoblot analysis of the effects of HA-CITED2 expression or pCPT-cAMP treatment (10 or 30 min) on phosphorylation of FLAG-GCN5 in AML12 cells as assessed with antibodies to phosphorylated PKA substrates. (c) Effects of CITED2 depletion on pCPT-cAMP-induced phosphorylation of FLAG-GCN5 and other PKA substrates as well as on the dephosphorylation of CRTC2 in primary hepatocytes. (d,e) IP and immunoblot analysis of the interaction of FLAG-GCN5 with Myc-PKAC and HA-CITED2 (d) as well as of the effect of CITED2 knockdown on the interaction of FLAG-GCN5 with PKAC (e) in AML12 cells. (f) Effect of siRNA-mediated PKAC depletion in AML12 cells on basal and CITED2-induced HAT activity of FLAG-GCN5 as assessed by in vitro assay. (g) Effect of PKAC overexpression in AML12 cells on HAT activity of FLAG-GCN5 measured in vitro. A Myc-PKAC plasmid and PKAC siRNA were introduced into cells by transfection, whereas adenoviral vectors were used to introduce other exogenous proteins or shRNAs in these experiments. Data are representative of at least three independent experiments. ANOVA, analysis of variance; siRNA; small interfering RNA.

Mentions: Given that recombinant CITED2 interacted with, but did not enhance the HAT activity of, GCN5 immunoprecipitated from AML12 cells overexpressing GCN5 alone (Supplementary Fig. 4a,b), we concluded that an additional factor that interacts with CITED2 might be necessary for the substrate switch. We found that, in the absence of pCPT-cAMP, inhibition of PKA by H89 significantly suppressed gluconeogenic gene induction by overexpression of PGC-1α alone or in combination with CITED2 in primary hepatocytes (Fig. 6a and Supplementary Fig. 4c), indicative of the requirement for PKA activity in this setting. PKA activated by glucagon-cAMP signalling mediates induction of gluconeogenesis and remodelling of the actin cytoskeleton through phosphorylation of CREB2627 and the inositol 1,4,5-trisphosphate receptor (IP3R)12 and through that of vasodilator-stimulated phosphoprotein (VASP)28, respectively. We then tested whether GCN5 activity is regulated by PKA-mediated phosphorylation. With the use of antibodies specific for phosphorylated PKA substrates, we found that GCN5 phosphorylation was induced by pCPT-cAMP in AML12 cells (Fig. 6b) as well as by glucagon in mouse liver (Supplementary Fig. 4d). This effect of pCPT-cAMP was enhanced by CITED2 overexpression (Fig. 6b) and suppressed by shRNA-mediated CITED2 knockdown (Fig. 6c). In contrast, CITED2 knockdown did not affect either the phosphorylation of other PKA substrates such as CREB, IP3R, and VASP or the dephosphorylation of CRTC2 (as assessed on the basis of the associated band mobility shift) induced by pCPT-cAMP (Fig. 6c). These results suggested that PKA selectively phosphorylates GCN5 in a CITED2-dependent manner, prompting us to examine whether PKA interacts with the GCN5-CITED2 complex.


The GCN5-CITED2-PKA signalling module controls hepatic glucose metabolism through a cAMP-induced substrate switch
GCN5 is phosphorylated by PKA within a GCN5-CITED2-PKA signalling module.(a) Effects of PKA inhibition with H89 (20 μM, 6 h) on gluconeogenic gene expression induced by overexpression of PGC-1α with or without CITED2 in primary hepatocytes. Data are means±s.e.m. (n=3). **P<0.01 (ANOVA with Bonferroni's post hoc test). (b) Immunoblot analysis of the effects of HA-CITED2 expression or pCPT-cAMP treatment (10 or 30 min) on phosphorylation of FLAG-GCN5 in AML12 cells as assessed with antibodies to phosphorylated PKA substrates. (c) Effects of CITED2 depletion on pCPT-cAMP-induced phosphorylation of FLAG-GCN5 and other PKA substrates as well as on the dephosphorylation of CRTC2 in primary hepatocytes. (d,e) IP and immunoblot analysis of the interaction of FLAG-GCN5 with Myc-PKAC and HA-CITED2 (d) as well as of the effect of CITED2 knockdown on the interaction of FLAG-GCN5 with PKAC (e) in AML12 cells. (f) Effect of siRNA-mediated PKAC depletion in AML12 cells on basal and CITED2-induced HAT activity of FLAG-GCN5 as assessed by in vitro assay. (g) Effect of PKAC overexpression in AML12 cells on HAT activity of FLAG-GCN5 measured in vitro. A Myc-PKAC plasmid and PKAC siRNA were introduced into cells by transfection, whereas adenoviral vectors were used to introduce other exogenous proteins or shRNAs in these experiments. Data are representative of at least three independent experiments. ANOVA, analysis of variance; siRNA; small interfering RNA.
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f6: GCN5 is phosphorylated by PKA within a GCN5-CITED2-PKA signalling module.(a) Effects of PKA inhibition with H89 (20 μM, 6 h) on gluconeogenic gene expression induced by overexpression of PGC-1α with or without CITED2 in primary hepatocytes. Data are means±s.e.m. (n=3). **P<0.01 (ANOVA with Bonferroni's post hoc test). (b) Immunoblot analysis of the effects of HA-CITED2 expression or pCPT-cAMP treatment (10 or 30 min) on phosphorylation of FLAG-GCN5 in AML12 cells as assessed with antibodies to phosphorylated PKA substrates. (c) Effects of CITED2 depletion on pCPT-cAMP-induced phosphorylation of FLAG-GCN5 and other PKA substrates as well as on the dephosphorylation of CRTC2 in primary hepatocytes. (d,e) IP and immunoblot analysis of the interaction of FLAG-GCN5 with Myc-PKAC and HA-CITED2 (d) as well as of the effect of CITED2 knockdown on the interaction of FLAG-GCN5 with PKAC (e) in AML12 cells. (f) Effect of siRNA-mediated PKAC depletion in AML12 cells on basal and CITED2-induced HAT activity of FLAG-GCN5 as assessed by in vitro assay. (g) Effect of PKAC overexpression in AML12 cells on HAT activity of FLAG-GCN5 measured in vitro. A Myc-PKAC plasmid and PKAC siRNA were introduced into cells by transfection, whereas adenoviral vectors were used to introduce other exogenous proteins or shRNAs in these experiments. Data are representative of at least three independent experiments. ANOVA, analysis of variance; siRNA; small interfering RNA.
Mentions: Given that recombinant CITED2 interacted with, but did not enhance the HAT activity of, GCN5 immunoprecipitated from AML12 cells overexpressing GCN5 alone (Supplementary Fig. 4a,b), we concluded that an additional factor that interacts with CITED2 might be necessary for the substrate switch. We found that, in the absence of pCPT-cAMP, inhibition of PKA by H89 significantly suppressed gluconeogenic gene induction by overexpression of PGC-1α alone or in combination with CITED2 in primary hepatocytes (Fig. 6a and Supplementary Fig. 4c), indicative of the requirement for PKA activity in this setting. PKA activated by glucagon-cAMP signalling mediates induction of gluconeogenesis and remodelling of the actin cytoskeleton through phosphorylation of CREB2627 and the inositol 1,4,5-trisphosphate receptor (IP3R)12 and through that of vasodilator-stimulated phosphoprotein (VASP)28, respectively. We then tested whether GCN5 activity is regulated by PKA-mediated phosphorylation. With the use of antibodies specific for phosphorylated PKA substrates, we found that GCN5 phosphorylation was induced by pCPT-cAMP in AML12 cells (Fig. 6b) as well as by glucagon in mouse liver (Supplementary Fig. 4d). This effect of pCPT-cAMP was enhanced by CITED2 overexpression (Fig. 6b) and suppressed by shRNA-mediated CITED2 knockdown (Fig. 6c). In contrast, CITED2 knockdown did not affect either the phosphorylation of other PKA substrates such as CREB, IP3R, and VASP or the dephosphorylation of CRTC2 (as assessed on the basis of the associated band mobility shift) induced by pCPT-cAMP (Fig. 6c). These results suggested that PKA selectively phosphorylates GCN5 in a CITED2-dependent manner, prompting us to examine whether PKA interacts with the GCN5-CITED2 complex.

View Article: PubMed Central - PubMed

ABSTRACT

Hepatic gluconeogenesis during fasting results from gluconeogenic gene activation via the glucagon&ndash;cAMP&ndash;protein kinase A (PKA) pathway, a process whose dysregulation underlies fasting hyperglycemia in diabetes. Such transcriptional activation requires epigenetic changes at promoters by mechanisms that have remained unclear. Here we show that GCN5 functions both as a histone acetyltransferase (HAT) to activate fasting gluconeogenesis and as an acetyltransferase for the transcriptional co-activator PGC-1&alpha; to inhibit gluconeogenesis in the fed state. During fasting, PKA phosphorylates GCN5 in a manner dependent on the transcriptional coregulator CITED2, thereby increasing its acetyltransferase activity for histone and attenuating that for PGC-1&alpha;. This substrate switch concomitantly promotes both epigenetic changes associated with transcriptional activation and PGC-1&alpha;&ndash;mediated coactivation, thereby triggering gluconeogenesis. The GCN5-CITED2-PKA signalling module and associated GCN5 substrate switch thus serve as a key driver of gluconeogenesis. Disruption of this module ameliorates hyperglycemia in obese diabetic animals, offering a potential therapeutic strategy for such conditions.

No MeSH data available.