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 训练营。相反，在肠道衍生的胰高血糖素样肽1（GLP1）中增加 通过促进胰岛素释放来增强葡萄糖清除。被认为是转录因子creb 介导两种肽激素的长期影响，在PKA磷酸化并与 CBP/P300。对营地的转录反应遵循爆发累积动力学； CREB活动达到峰值 1小时的刺激，4-6小时后返回基线。 除了对CREB磷酸化的影响外，胰高血糖素和GLP1还通过 刺激其与cAMP调节的转录共激活因子（CRTC/TORC）的关联，潜在 细胞质CREB辅助因子，其去磷酸化后，将其转移到核，以响应其去磷酸化。 营。 CRTC1仅在大脑中表达，而CRTC2和CRTC3在大多数组织中共表达。这 CRTC2和CRTC3在重叠或CREB靶基因的不同子集中起作用的程度是 但是，不清楚。在上一个赠款期间，我们表明CREB/CRTC2途径有助于 重要的是禁食葡萄糖生产； CRTC2在肝脏中的急性耗竭大大降低了血糖 浓度和糖原性基因表达，而野生型的过表达和更大程度的过表达 磷酸化缺陷CRTC2增加了糖异生。 与急性肝CRTC2敲低的影响相反，CRTC2的全身敲除小鼠 仅显示禁食葡萄糖水平的适度降低；他们会在他们发展时出现胰岛素分泌缺陷 年龄。这些结果表明，其他CREB共激活因子涉及，以补偿 肝脏中的CRTC2，他们建议胰岛中的CRTC2表达也可以调节循环 葡萄糖浓度通过其对胰岛素分泌的影响。支持后者，MAFA，一个Beta细胞 胰岛素分泌所需的转录因子被CREB和CRTC2强烈上调。 在即将到来的赠款期间的拟议研究重点是CRTC成员的假设 家庭对CREB活动产生重叠的影响。新确定的Creb互动的重要性 在CREB活性增强的蛋白质和CRTC2突变小鼠中CRTC2损失的补偿将是 测试。最后，有效的Creb抑制剂的作用，该抑制剂在胰岛下的上调 将评估高血糖条件，以促进对GS耦合受体信号传导的抗性。 提出了三个目标；他们通过解决Creb的机制来扩展先前的工作 途径促进肝脏中的糖异生，并促进胰岛中的胰岛素分泌。 在AIM 1中，我们将使用与CRTC2和CRTC3的Floxed等位基因的小鼠评估这些相对作用 调节肝糖异生和胰岛素分泌方面的共激活剂。我们将与组织产生小鼠 肝脏或胰岛中CRTC2和CRTC3的特定敲除。做CRTC2和CRTC3施加 对肝脏中糖原基因表达的重叠作用？他们是否促进胰岛素分泌 上调亮氨酸拉链因子MAFA？ 在AIM 2中，我们将测试在CREB蛋白质组学中鉴定的BRD2-A溴ab蛋白的作用 相关蛋白质 - 刺激糖原性基因的表达。我们将在BRD2中表征域 和介导这种相互作用的creb； Creb乙酰化在调节BRD2：CREB中的作用 协会也将进行测试。我们将通过给药来评估BRD2的抑制 选择性溴结构域抑制剂，在胰岛素抵抗的情况下改善了葡萄糖水平。 在AIM 3中，我们将研究胰岛中CREB靶基因表达的机制 在胰岛素抵抗中下调。特别是，我们将研究蛋白激酶抑制剂β的作用 （PKIB）在干扰GLP1和其他激素后，其上调响应高血糖： PKIB敲除小鼠将用于确定该抑制剂的耗尽是否改善胰岛 在胰岛素抵抗的情况下的功能。 综上所述，拟议的研究将提供有关胰高血糖素和胰高血糖素和 GLP1通过对肝脏和胰腺β细胞中CREB途径的影响来促进葡萄糖平衡。 进度报告 具体目标1：我们将检查组蛋白乙酰基转移酶PSOO和CBP（P300/CBP）和 NAD+依赖性脱乙酰基酶SIRTI通过乙酰化和脱乙酰化调节T0RC2活性 在禁食期间。我们将确定经历乙酰化的T0RC2（CRTC2）中的残留物，我们将测试 p300/cbp和sirt1在此过程中的重要性通过肝过表达或每种蛋白质的耗竭。 乙酰化在通过蛋白质稳定增强T0RC2活性中的重要性也将是 解决。 我们发现CBP/P300在某种程度上通过促进CRTC2乙酰化来增强了TORC2/CRTC2的活性 Lys628，在CRTC家庭成员中保守的残留物（1）。相反，发现Sirti 通过在lys628处通过crtc2脱乙酰化，可以部分抑制CRTC2活性。 LYS628似乎调节CRTC2 稳定。响应cAPC信号的核进入后，CRTC2在 LYS628通过与CO P I的关联，CO P I是CUL4A E3连接酶的适配器组件。当它退出核时， 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 在Lys136（2）处，CREB本身的乙酰化 /脱乙酰化。探索基本机制，我们有 开发了一种乙酰溶解酶136特异性CREB抗血清。 CREB乙酰化在SIRTI - / - 组成构成上调 小鼠胚胎成纤维细胞，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在与之相关并增强的潜在作用 溶液36-乙酰化的Creb的活性。 具体目的2：我们将研究盐诱导激酶（SIKS）在调节肝T0RC2中的作用 （CRTC2）通过磷酸化p300/cbp并抑制其与T0RC2的关联来活性。我们将确定 p300/cbp中的SIK2磷酸化位点，我们将测试SIK2在催化P300/CBP中的重要性 通过过表达或RNAi介导的Sik2在肝脏中的耗竭来磷酸化。 p300/cbp的作用 调节肝T0RC2活性中的磷酸化也将使用磷酸化缺陷型确定 PSOO MTITANT蛋白。 14-3-3蛋白在与磷酸化的PSO0/CBP和 从而破坏TORC2：P300/CBP相互作用也将进行检查。 发现SIK2在Ser89（以PSOO）（1）磷酸化CBP和PSOO磷酸化。反过来Ser89磷酸化 暴露于胰高血糖素的肝细胞中CREB靶基因上的CBP/PSOO活性降低。反过来， Ser89Ala突变体PSOO比野生型PSOO更活跃，在支持CREB依赖转录方面。 尽管Ser89构成了14-S-S结合共识基序的一部分，但我们无法检测到任何关联 CBP或PSOO的肝细胞或其他细胞中的14-3-S蛋白。 除了它们对CBP/PSOO的影响外，我们还发现Siks还调节了ILA HDAC类的活动 在肝脏中（3，4）。 ILA类HDAC在基础条件下通过磷酸化在细胞核中被隔离 与14-S-3蛋白的相互作用相互作用；当他们响应营地激动剂时，他们搬到了核心 PKA依赖性磷酸化抑制了SIK。虽然在骨骼肌中首次识别 在包括肝脏在内的大多数细胞类型中刺激ILA类HDAC类的易位。确实，核的增加 禁食过程中的ILA hDAC类似乎促进了肝糖生成，部分通过去乙酰化来促进 f o x 0 1。在拟议的研究的目标1中，我们将研究ILA类HDAC途径是否 补偿肝脏中CRTC2和CRTCs表达的损失。 特定目标3：我们将研究T0RC2（CRTC2）在触发糖原的过程中的作用 在与组蛋白甲基转移酶（HMT）复合物的缔合过程中禁食期间。我们将监视组蛋白 甲基化对糖原性基因，我们将评估T0RC2在介导的重要性 通过RNAi介导的耗竭或突变torc2蛋白过表达的过程，这些蛋白有缺陷 HMT相互作用。 HMT在通过甲基化调节糖原基因表达中的潜在作用 T0RC2也将进行研究。 我们发现CRTC2/TORC2与WDR5（组蛋白甲基转移酶的核心成分）相关联 （HMT）复合物（5）。 RNAi介导的WDR5敲低可降低糖原基因上的CRTC2活性 在暴露于胰高血糖素的细胞中；但是敲低其他核心组件，例如ASH2L和RBBP4没有 尽管HSK4三甲基化在ASH2L或 RBBP4耗尽细胞。相反，我们发现WDR5通过调节调节糖原基因表达 组蛋白乙酰转移酶配合物的活性，其中包含旁系同源物GCN5（KAT2A）和PCAF （KAT2B）。 实际上，kat2a/b直接与CRTC2的反式激活结构域（TAD）相关（5）；和突变