Regulation of Hepatic Gluconeogenesis by the CREB:TORC2 Pathway

CREB:TORC2 通路对肝糖异生的调节

• 批准号: 8749897
• 负责人: MARC R MONTMINY
• 金额: $ 75.55万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-07 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8749897
• 关键词: 14-3-3 Proteins; Acetylation; Acute; Address; Age; Agonist; BRD2 gene; Beta Cell; Binding; Blood Glucose; Brain; Bromodomain; CREB1 gene; Cell Nucleus; Cells; Complex; Consensus; Coupled; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Deacetylase; Deacetylation; Defect; EP300 gene; Embryo; Equilibrium; Family; Family member; Fasting; Fibroblasts; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Glucagon; Gluconeogenesis; Glucose; Grant; Hepatic; Hepatocyte; Histone Deacetylase; Histones; Hormones; Hour; Hyperglycemia; Immune Sera; Insulin; Insulin Resistance; Islets of Langerhans; Kinetics; Knock-out; Knockout Mice; Leucine Zippers; Liver; Long-Term Effects; LoxP-flanked allele; Mediating; Methylation; Monitor; Mono-S; Mus; Mutant Strains Mice; Mutation; Nuclear; PCAF gene; Pathway interactions; Phosphorylation; Phosphorylation Site; Process; Progress Reports; Protein Dephosphorylation; Protein Kinase Inhibitors; Protein S; Proteins; Proteomics; RNA Interference; Receptor Signaling; Regulation; Relative (related person); Resistance; Role; Signal Transduction; Skeletal Muscle; Structure of beta Cell of islet; Testing; Tissues; Transcription Coactivator; Transferase; Ubiquitination; Up-Regulation; Work; abstracting; attenuation; cell type; cofactor; fasting glucose; feeding; glucagon-like peptide; glucose production; hepatic gluconeogenesis; improved; inhibitor/antagonist; insight; insulin secretion; member; multicatalytic endopeptidase complex; mutant; pancreatic islet function; paralogous gene; peptide hormone; programs; protein kinase inhibitor; response; salt-inducible kinase; transcription factor; ubiquitin-protein ligase

Abstract Under fasting conditions, increases in circulating glucagon stimulate hepatic glucose production via induction ofthe cAMP pathway. Conversely, increases in gut-derived glucagon-like peptide 1 (GLP1) during feeding enhance glucose clearance by promoting insulin release. The transcription factor CREB is thought to mediate long term effects of both peptide hormones, following its phosphorylation by PKA and association with CBP/P300. The transcriptional response to cAMP follows burst-attenuation kinetics; CREB activity peaks after 1 hour of stimulation, returning to baseline after 4-6 hours. In addition to their effects on CREB phosphorylation, glucagon and GLP1 also increase CREB activity by stimulating its association with the cAMP Regulated Transcriptional Coactivators (CRTCs/TORCs), latent cytoplasmic CREB cofactors that translocate to the nucleus following their dephosphorylation in response to cAMP. CRTC1 is expressed only in brain, while CRTC2 and CRTC3 are co-expressed in most tissues. The extent to which CRTC2 and CRTC3 function on overlapping or distinct subsets of CREB target genes is unclear, however. In the previous grant period, we showed that the CREB/CRTC2 pathway contributes importantly to fasting glucose production; acute depletion of CRTC2 in liver substantially lowers blood glucose concentrations and gluconeogenic gene expression, while over-expression of wild-type and to a greater extent phosphorylation-defective CRTC2 increases gluconeogenesis. By contrast with effects of acute hepatic CRTC2 knockdown, mice with a whole-body knockout of CRTC2 show only modest reductions in fasting glucose levels; and they develop an insulin secretion defect as they age. These results point to the involvement of additional CREB coactivators that compensate for loss of CRTC2 in liver, and they suggest that CRTC2 expression in pancreatic islets also modulates circulating glucose concentrations through its effects on insulin secretion. Supporting the latter, MafA, a beta cell transcription factor that is required for insulin secretion, is strongly upregulated by CREB and CRTC2. Proposed studies during the upcoming grant period focus on the hypothesis that members ofthe CRTC family exert overlapping effects on CREB activity. The importance of a newly identified CREB interacting protein in potentiating CREB activity and compensating for loss of CRTC2 in CRTC2 mutant mice will be tested. Finally the role of a potent CREB inhibitor, which is upregulated in pancreatic islets under hyperglycemic conditions, in promoting resistance to Gs-coupled receptor signaling, will be evaluated. Three aims are proposed; they extend the previous work by addressing the mechanisms by which the CREB pathway promotes gluconeogenesis in liver and facilitates insulin secretion from pancreatic islets. In Aim 1, we will use mice with floxed alleles of CRTC2 and CRTC3 to evaluate the relative roles of these coactivators in modulating hepatic gluconeogenesis and insulin secretion. We will generate mice with tissue ' specific knockouts of CRTC2 and CRTC3 in liver or pancreatic islets. Do CRTC2 and CRTC3 exert overlapping effects on gluconeogenic gene expression in liver? Do they promote insulin secretion by upregulating the leucine zipper factor MafA? In Aim 2, we will test the role of BRD2-a bromodomain protein identified in a proteomic screen for CREB associated proteins- in stimulating expression of gluconeogenic genes. We will characterize domains in BRD2 and CREB that mediate this interaction; and the role of CREB acetylation in modulating the BRD2:CREB association will also be tested. We will evaluate whether inhibition of BRD2, through administration of a selective bromodomain inhibitor, improves glucose levels in the setting of insulin resistance. In Aim 3, we will examine the mechanism by which CREB target gene expression in pancreatic islets is down-regulated in insulin resistance. In particular, we will investigate the role of Protein Kinase Inhibitor beta (PKIB) in interfering with GLP1 and other hormones, following its upregulation in response to hyperglycemia: PKIB knockout mice will be used to determine whether depletion of this inhibitor improves pancreatic islet function in the setting of insulin resistance. Taken together, the proposed studies will provide new insight into mechanisms by which glucagon and GLP1 promote glucose balance through their effects on the CREB pathway in liver and pancreatic beta cells. Progress Report Specific Aim 1: We will examine the role ofthe histone acetyl-transferases PSOO and CBP (P300/CBP) and the NAD+ dependent deacetylase SIRTI in modulating T0RC2 activity through acetylation and deacetylation during fasting. We will identify residues in T0RC2 (CRTC2) that undergo acetylation, and we will test the importance of P300/CBP and SIRT1 in this process by hepatic over-expression or depletion of each protein. The importance of acetylation in augmenting T0RC2 activity through protein stabilization will also be addressed. We found that CBP/P300 enhanced TORC2/CRTC2 activity in part by promoting CRTC2 acetylation at Lys628, a residue that is well conserved amongst CRTC family members (1). Conversely, SirTI was found to inhibit CRTC2 activity in part through deacetylation of CRTC2 at Lys628. Lys628 appears to regulate CRTC2 stability. Following its nuclear entry in response to cAMP signaling, CRTC2 undergoes mono-ubiquitination at Lys628 via an association with C O P I , the adaptor component of a Cul4A E3 ligase. When it exits the nucleus, mono-ubiquitinated CRTC2 undergoes poly-ubiquitination and proteasome-mediated degradation. In addition to their effects on CRTC2, CBP and SirTI also appear to regulate CREB target genes through acetylation /deacetylation of CREB itself at Lys136 (2). To explore the underlying mechanism, we have developed an acetyl-lys136 specific CREB antiserum. CREB acetylation is constitutively upregulated in SirTI-/- mouse embryo fibroblasts and it is absent in CBP-/-, P300-/- mutant cells. In Aim 2 ofthe proposed studies, we will address the potential role of the bromodomain coactivator BRD2 in associating with and potentiating the activity of Lysl 36-acetylated CREB. Specific Aim 2: We will investigate the role of Salt Inducible Kinases (SIKs) in modulating hepatic T0RC2 (CRTC2) activity by phosphorylating P300/CBP and inhibiting their association with T0RC2. We will identify SIK2 phosphorylation sites in P300/CBP, and we will test the importance of SIK2 in catalyzing P300/CBP phosphorylation by over-expression or RNAi mediated depletion of SIK2 in liver. The role of P300/CBP phosphorylation in modulating hepatic T0RC2 activity will also be determined using phosphorylation-defective PSOO mtitant proteins. The potential role of 14-3-3 proteins in binding to phosphorylated PSO0/CBP and thereby disrupting the TORC2:P300/CBP interaction will also be examined. SIK2 was found to phosphorylate CBP and PSOO at Ser89 (in PSOO) (1). In turn Ser89 phosphorylation reduced CBP/PSOO activity over CREB target genes in hepatocytes exposed to glucagon. Conversely, Ser89Ala mutant PSOO was more active than wild-type PSOO in supporting CREB dependent transcription. Although Ser89 forms part of a consensus motif for 14-S-S binding, we were unable to detect any association of either CBP or PSOO with 14-3-S proteins in hepatocytes or other cells. In addition to their effects on CBP/PSOO, we found that SIKs also regulate the activities of class Ila HDACs in liver (3, 4). Class Ila HDACs are sequestered in the nucleus under basal conditions through phosphorylation dependent interactions with 14-S-3 proteins; and they move to the nucleus in response to cAMP agonist, when SIKS are inhibited by PKA-dependent phosphorylation. Although first identified in skeletal muscle, cAMP stimulates the translocation of Class Ila HDACs in most cell types, including liver. Indeed, increases in nuclear class Ila HDACs during fasting appears to promote hepatic gluconeogenesis, in part through the de-acetylation of F O X 0 1 . In Aim 1 of the proposed studies, we will investigate whether the class Ila HDAC pathway compensates for loss of CRTC2 and CRTCS expression in liver. Specific Aim 3: We will investigate the role of T0RC2 (CRTC2) in triggering the gluconeogenic program during fasting through its association with a histone methyl transferase (HMT) complex. We will monitor histone methylation over gluconeogenic genes, and we will evaluate the importance of T0RC2 in mediating this process through RNAi mediated depletion or over-expression of mutant TORC2 proteins that are defective in the HMT interaction. The potential role of HMTs in modulating gluconeogenic gene expression by methylating T0RC2 will also be investigated. We found that CRTC2/TORC2 associates with WDR5, a core component of histone methyl transferase (HMT) complexes (5). RNAi-mediated knockdown of WDR5 reduces CRTC2 activity over gluconeogenic genes in cells exposed to glucagon; but knockdown of other core components such as Ash2l and RbbP4 had no effect, on gluconeogenic gene expression, despite substantial reductions in HSK4 tri-methylation in Ash2l or RbBp4 depleted cells. Rather we found that WDR5 regulates gluconeogenic gene expression by modulating the activity of histone acetyl transferase complexes, which contain the paralogs GCN5 (KAT2A) and PCAF (KAT2B). Indeed, KAT2A/B associate directly with the trans-activation domain (TAD) of CRTC2 (5); and mutations'in

抽象的 在禁食条件下，循环胰高血糖素的增加通过以下方式刺激肝葡萄糖产生： cAMP 途径的诱导。相反，肠道来源的胰高血糖素样肽 1 (GLP1) 的增加 喂养通过促进胰岛素释放来增强葡萄糖清除率。转录因子 CREB ​​被认为 介导两种肽激素的长期作用，通过 PKA 磷酸化并与 CBP/P300。对 cAMP 的转录反应遵循突发衰减动力学； CREB ​​活动达到峰值后 刺激 1 小时，4-6 小时后恢复基线。 除了对 CREB ​​磷酸化的影响外，胰高血糖素和 GLP1 还可以通过以下方式增加 CREB ​​活性： 刺激其与 cAMP 调节转录辅激活因子 (CRTC/TORC) 的关联，潜在 细胞质 CREB ​​辅因子在响应去磷酸化后转位至细胞核 营。 CRTC1仅在脑中表达，而CRTC2和CRTC3在大多数组织中共表达。这 CRTC2 和 CRTC3 在 CREB ​​靶基因的重叠或不同子集上发挥作用的程度 然而还不清楚。在之前的资助期间，我们表明 CREB/CRTC2 途径有助于 对空腹血糖产生很重要；肝脏中 CRTC2 的急性消耗显着降低血糖 浓度和糖异生基因表达，而野生型的过度表达和更大程度 磷酸化缺陷的 CRTC2 会增加糖异生。 与急性肝脏 CRTC2 敲除的效果相比，全身 CRTC2 敲除的小鼠 空腹血糖水平仅略有下降；他们会出现胰岛素分泌缺陷 年龄。这些结果表明额外的 CREB ​​共激活剂的参与弥补了 CRTC2 在肝脏中，他们表明 CRTC2 在胰岛中的表达也调节循环 葡萄糖浓度通过其对胰岛素分泌的影响。支持后者的是 MafA，一种 β 细胞 CREB ​​和 CRTC2 强烈上调胰岛素分泌所需的转录因子。 在即将到来的资助期内拟议的研究重点是 CRTC 成员的假设 家庭对 CREB ​​活性产生重叠影响。新发现的 CREB ​​相互作用的重要性 增强CREB活性并补偿CRTC2突变小鼠中CRTC2缺失的蛋白质将被 已测试。最后是一种有效的 CREB ​​抑制剂的作用，它在胰岛中的表达上调。 将评估高血糖条件下促进 Gs 偶联受体信号传导抵抗的情况。 提出了三个目标；他们通过解决 CREB ​​所使用的机制来扩展之前的工作 途径促进肝脏中的糖异生并促进胰岛的胰岛素分泌。 在目标 1 中，我们将使用具有 CRTC2 和 CRTC3 等位基因 floxed 的小鼠来评估这些基因的相对作用 调节肝糖异生和胰岛素分泌的共激活剂。我们将用组织培育小鼠' 肝或胰岛中 CRTC2 和 CRTC3 的特异性敲除。 CRTC2和CRTC3发挥作用吗 对肝脏糖异生基因表达的重叠影响？它们通过以下方式促进胰岛素分泌吗 上调亮氨酸拉链因子 MafA？ 在目标 2 中，我们将测试 BRD2（一种在 CREB ​​蛋白质组筛选中鉴定出的溴结构域蛋白）的作用 相关蛋白-刺激糖异生基因的表达。我们将表征 BRD2 中的域 和 CREB ​​调解这种相互作用；以及 CREB ​​乙酰化在调节 BRD2:CREB ​​中的作用 关联也将受到测试。我们将评估是否通过给予 BRD2 进行抑制 选择性溴结构域抑制剂，可改善胰岛素抵抗情况下的血糖水平。 在目标 3 中，我们将研究胰岛中 CREB ​​靶基因表达的机制 胰岛素抵抗下调。特别是，我们将研究蛋白激酶抑制剂 β 的作用 (PKIB) 干扰 GLP1 和其他激素，其因高血糖而上调： PKIB 敲除小鼠将用于确定消除该抑制剂是否会改善胰岛 在胰岛素抵抗的情况下发挥作用。 总而言之，拟议的研究将为胰高血糖素和 GLP1 通过影响肝脏和胰腺 β 细胞中的 CREB ​​通路来促进葡萄糖平衡。 进度报告 具体目标 1：我们将研究组蛋白乙酰转移酶 PSOO 和 CBP (P300/CBP) 的作用以及 NAD+ 依赖性脱乙酰酶 SIRTI 通过乙酰化和脱乙酰化调节 TORC2 活性 禁食期间。我们将鉴定 T0RC2 (CRTC2) 中经历乙酰化的残基，并测试 通过肝脏过度表达或每种蛋白质的消耗，P300/CBP 和 SIRT1 在此过程中的重要性。 乙酰化在通过蛋白质稳定增强 T0RC2 活性方面​​的重要性也将是 已解决。 我们发现 CBP/P300 部分通过促进 CRTC2 乙酰化来增强 TORC2/CRTC2 活性 Lys628，CRTC 家族成员中高度保守的残基 (1)。相反，SirTI 被发现 部分通过 CRTC2 Lys628 脱乙酰化来抑制 CRTC2 活性。 Lys628 似乎调节 CRTC2 稳定。 CRTC2 响应 cAMP 信号进入核后，在以下位置经历单泛素化： Lys628 通过与 COPI（Cul4A E3 连接酶的衔接子组件）关联。当它离开细胞核时， 单泛素化的 CRTC2 经历多泛素化和蛋白酶体介导的降解。 除了对 CRTC2 的影响外，CBP 和 SirTI 似乎还通过以下方式调节 CREB ​​靶基因： CREB ​​本身在 Lys136 处的乙酰化/脱乙酰化 (2)。为了探究其背后的机制，我们有 开发了乙酰-lys136特异性CREB抗血清。 SirTI-/- 中 CREB ​​乙酰化持续上调 小鼠胚胎成纤维细胞中存在，并且在 CBP-/-、P300-/- 突变细胞中不存在。在拟议研究的目标 2 中，我们 将解决溴结构域共激活剂 BRD2 在关联和增强 Lysl 36-乙酰化 CREB ​​的活性。 具体目标 2：我们将研究盐诱导激酶 (SIK) 在调节肝 T0RC2 中的作用 (CRTC2) 活性通过磷酸化 P300/CBP 并抑制其与 T0RC2 的关联来实现。我们将确定 SIK2在P300/CBP中的磷酸化位点，我们将测试SIK2在催化P300/CBP中的重要性 肝脏中 SIK2 的过度表达或 RNAi 介导的耗竭导致磷酸化。 P300/CBP 的作用 调节肝 T0RC2 活性的磷酸化也将使用磷酸化缺陷来确定 PSOO 突变蛋白。 14-3-3 蛋白在结合磷酸化 PSO0/CBP 和 还将检查由此破坏 TORC2:P300/CBP 相互作用的情况。 人们发现 SIK2 在 Ser89（PSOO 中）处磷酸化 CBP 和 PSOO (1)。 Ser89 依次磷酸化 暴露于胰高血糖素的肝细胞中 CBP/PSOO 活性相对于 CREB ​​靶基因降低。反过来， Ser89Ala 突变体 PSOO 在支持 CREB ​​依赖性转录方面比野生型 PSOO 更活跃。 尽管 Ser89 构成 14-S-S 结合的共有基序的一部分，但我们无法检测到任何关联 CBP 或 PSOO 与肝细胞或其他细胞中的 14-3-S 蛋白的结合。 除了对 CBP/PSOO 的影响外，我们发现 SIK 还调节 Ila 类 HDAC 的活性 在肝脏中 (3, 4)。 IIa 类 HDAC 在基础条件下通过磷酸化被隔离在细胞核中 与 14-S-3 蛋白的依赖性相互作用；它们响应 cAMP 激动剂而移动到细胞核，当 SIKS 受到 PKA 依赖性磷酸化的抑制。尽管 cAMP 首次在骨骼肌中被发现， 刺激大多数细胞类型（包括肝脏）中 IIa 类 HDAC 的易位。事实上，核电的增加 禁食期间的IIa类HDAC似乎部分地通过脱乙酰化促进肝糖异生 的FO X 0 1 。在拟议研究的目标 1 中，我们将研究 IIa 类 HDAC 通路是否 补偿肝脏中 CRTC2 和 CRTCS 表达的损失。 具体目标 3：我们将研究 T0RC2 (CRTC2) 在触发糖异生程序中的作用 禁食期间通过其与组蛋白甲基转移酶（HMT）复合物的关联。我们将监测组蛋白 糖异生基因的甲基化，我们将评估 T0RC2 在介导这一过程中的重要性 通过 RNAi 介导的突变型 TORC2 蛋白的缺失或过度表达来实现这一过程，这些突变型 TORC2 蛋白在 HMT 相互作用。 HMT 通过甲基化调节糖异生基因表达的潜在作用 T0RC2也将受到调查。 我们发现 CRTC2/TORC2 与组蛋白甲基转移酶的核心成分 WDR5 相关 (HMT) 复合物 (5)。 RNAi 介导的 WDR5 敲低降低了 CRTC2 对糖异生基因的活性 在暴露于胰高血糖素的细胞中；但其他核心成分如 Ash2l 和 RbbP4 的敲除却没有 尽管 Ash2l 或 HSK4 三甲基化大幅减少，但对糖异生基因表达的影响 RbBp4 耗尽的细胞。相反，我们发现 WDR5 通过调节糖异生基因表达 组蛋白乙酰转移酶复合物的活性，其中包含旁系同源物 GCN5 (KAT2A) 和 PCAF （KAT2B）。 事实上，KAT2A/B 直接与 CRTC2 的反式激活结构域 (TAD) 关联 (5)；和突变