Spatiotemporal control of insulin signaling by mitotic regulators

有丝分裂调节剂对胰岛素信号传导的时空控制

• 批准号: 10668524
• 负责人: Eunhee Choi
• 金额: $ 41.13万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-20 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10668524
• 关键词: Affect; Animal Model; Animals; Attenuated; Binding; Biochemistry; Cell division; Cell membrane; Cell surface; Cells; Cellular biology; Chemicals; Clathrin; Clathrin Adaptors; Complex; Cryoelectron Microscopy; Cultured Cells; DNA Sequence Alteration; Data; Defect; Diabetes Mellitus; Diet; Disease; Endocytosis; Endoplasmic Reticulum; Ensure; FRAP1 gene; Feedback; Genome Stability; Genomics; Goals; Growth; Hepatic; Homeostasis; Human; Insulin; Insulin Receptor; Insulin Resistance; Insulin Signaling Pathway; Insulin deficiency; Knock-out; Lead; Link; Lipids; Liver; MAP Kinase Gene; MXI1 gene; Mediating; Mediator; Metabolic; Metabolic Control; Metabolic Diseases; Metabolism; Mitosis; Mitotic; Molecular Target; Mus; Non-Insulin-Dependent Diabetes Mellitus; PIK3CG gene; PTPN1 gene; PTPN11 gene; Pathogenesis; Pathway interactions; Persons; Phosphorylation; Phosphotransferases; Physiological; Physiology; Protein Tyrosine Phosphatase; Proto-Oncogene Proteins c-akt; Quality Control; Receptor Signaling; Regulation; Research; Resistance; Role; Signal Pathway; Signal Transduction; Structure; Testing; blood glucose regulation; clinical application; clinical development; enhancer-binding protein AP-2; glucose metabolism; improved; in vivo; inhibitor; innovation; insulin regulation; insulin sensitivity; insulin signaling; lipid biosynthesis; lipid metabolism; metabolic phenotype; mouse genetics; mutant; new therapeutic target; receptor; recruit; spatiotemporal; trafficking

SPECIFIC AIMS: Precise regulation of the insulin signaling pathway is critical for multiple facets of animal physiology 1-3. Dysregulation of the insulin signaling pathway has been linked to metabolic disorders, such as diabetes 4. As type 2 diabetes affects more than 400 million people worldwide 5, understanding the signaling pathways impacting this disease is of paramount importance. Genetic mutations of the insulin receptor (IR) cause rare and severe insulin resistance 6. Yet, the causes of insulin resistance seen in type 2 diabetes are numerous and the mechanisms are multifactorial leaving many unanswered questions. Upon insulin binding at the plasma membrane (PM), IR triggers the activation of bifurcated signaling pathways: the PI3K-AKT pathway for metabolism and the MAPK pathway for growth. Active IR is then internalized by clathrin-mediated endocytosis 7. The IR endocytosis has been extensively studied for decades 7. Yet, how cell surface levels of functional IR in the basal and insulin-stimulated states are regulated, how IR trafficking affects the activation of specific signaling pathway in vivo, and how dysregulation of IR trafficking contributes to human insulin resistance remain largely unclear. Answering these questions requires identifying specific mediators and regulators of IR endocytosis, and a mechanistic understanding of the IR endocytic pathways in an animal model. Our long-term goal is to understand how systemic IR signaling controls metabolic homeostasis and genome stability. Our recent studies show that MAD2, a key mitosis regulator, cooperates with IR substrate (IRS) to promote IR endocytosis through the recruitment of the clathrin adaptor complex AP2 to the IR 8-10. Mechanistically, MAD2 constitutively binds to the IR through a well-conserved MAD2-interacting motif (MIM). The MAD2 inhibitor p31comet blocks the MAD2-dependent AP2 recruitment to IR. A phosphorylation switch of IRS controlled by MAPK and the tyrosine phosphatase SHP2 ensures selective internalization of insulin-activated IR. Targeting this feedback regulation prolongs the metabolic branch of IR signaling and improves insulin sensitivity in mice. Our preliminary data show that IR4A/4A mice (deficient for MAD2-binding and endocytosis) are resistant to diet-induced insulin resistance. We found that MAD2 is also required to keep ATP-binding-deficient IR (kinase dead) mutants in the endoplasmic reticulum (ER). We hypothesize that mitotic regulators maintain metabolic homeostasis by exerting spatiotemporal control of IR signaling inside the cell. To test this, we will: AIM 1. Establish the physiological function of IR spatiotemporal control by MAD2. Our preliminary data show that IR4A/4A mice, in which IR cannot bind to MAD2, display delayed IR endocytosis and prolonged IR signaling. We hypothesize that IR trafficking by MAD2 controls glucose and lipid metabolism. We will analyze the metabolic phenotypes of IR4A/4A mice, using wild-type (WT), liver-specific-p31-/- (liver-p31-/-), and liver-IR-/- mice as controls. We will also test the role of IR spatiotemporal control in metabolism using chemical inhibitors of SHP2 in mice and cultured cells. AIM 2. Determine the mechanism by which IR-MAD2 binding promotes hepatic lipogenesis. mTOR complex (mTORC) promotes lipid synthesis through activation of SREBP1 in the liver 11,12. mTORC1 also attenuates IR signaling by negative feedback loops 13-17. Our preliminary data show that IR4A/4A mice display hepatic defects in SREBP1 activation and de novo lipogenesis, although the PI3K-AKT pathway (an important activator of mTORC1) is enhanced in the liver. We hypothesize that IR trafficking by MAD2 promotes hepatic lipogenesis through the mTORC-SREBP1 pathway or through the negative feedback loops. We will test if endocytosed IR can still retain partial activity, and signal locally to promote lipogenesis. If so, then we will identify molecular targets of the endocytosed IR kinase required to control lipogenesis in the liver. We will also examine the role of IR-MAD2 binding in the mTORC1-dependent negative feedback loops in both mice and cultured cells. AIM 3. Elucidate the mechanism of quality control and trafficking of IR by MAD2 and PTP1B. The receptor tyrosine phosphatase PTP1B is implicated in the regulation of IR signaling at the PM and in the ER 18-22. Our preliminary results show that an ATP-binding-deficient IR mutant, but not catalytic dead IR mutants, is retained in the ER. Strikingly, both disruption of the IR-MAD2 interaction and knockout of PTP1B lead to release the ATP- binding-deficient IR mutant from the ER. We hypothesize that PTP1B and MAD2 increase functional IR levels at the PM by sequestering ATP-binding-deficient IR in the ER. We will probe whether MAD2 and p31comet facilitate IR-PTP1B interaction in the ER and promote degradation of the retained IR. If so, then we will determine how MAD2-PTP1B controls such degradation. By determining the structure of PTP1B-IR-MAD2-p31comet complex by cryo-EM, we will elucidate the mechanism by which MAD2-PTP1B regulates IR quality control and trafficking. Our innovative approach combining mouse genetics, cell biology, biochemistry, cryo-EM, and genomics to investigate the roles of mitotic regulators in metabolic homeostasis will advance our understanding of the regulatory mechanism(s) governing spatiotemporal IR signaling. Importantly, both the IR signaling and the spindle checkpoint machinery are highly conserved from mice to hu lts will aid in the development of clinical applications for the treatment of type 2 diabetes.

具体目标：胰岛素信号通路的精确调节对于动物生理学的多个方面至关重要1-3。胰岛素信号通路的失调与代谢紊乱有关，如4型糖尿病。由于2型糖尿病影响着全球4亿多人，因此了解影响这种疾病的信号通路至关重要。胰岛素受体（IR）的基因突变导致罕见和严重的胰岛素抵抗6。然而，在2型糖尿病中观察到的胰岛素抵抗的原因很多，其机制是多因素的，留下许多未回答的问题。 当胰岛素在质膜（PM）处结合时，IR触发分叉信号传导途径的激活：用于代谢的PI 3 K-AKT途径和用于生长的MAPK途径。然后通过网格蛋白介导的内吞作用将活性IR内化7。IR内吞作用已经被广泛研究了几十年7。然而，在基础状态和胰岛素刺激状态下，细胞表面功能性IR水平是如何调节的，IR运输如何影响体内特定信号传导途径的激活，以及IR运输的失调如何导致人胰岛素抵抗仍然在很大程度上不清楚。解决这些问题需要确定特定的介质和调节IR内吞作用，并在动物模型中的IR内吞途径的机械理解。 我们的长期目标是了解全身IR信号如何控制代谢稳态和基因组稳定性。我们最近的研究表明，MAD 2，一个关键的有丝分裂调节剂，与IR底物（IRS）合作，通过招募网格蛋白适配器复合物AP 2到IR 8-10来促进IR内吞作用。从机制上讲，MAD 2通过一个保守的MAD 2相互作用基序（MIM）与IR组成性结合。MAD 2抑制剂p31 comet阻断MAD 2依赖的AP 2向IR的募集，MAPK和酪氨酸磷酸酶SHP 2控制的IRS磷酸化开关确保胰岛素激活的IR选择性内化，靶向这种反馈调节阻断IR信号的代谢分支，改善小鼠胰岛素敏感性。我们的初步数据表明，IR 4A/4A小鼠（缺乏MAD 2结合和内吞作用）对饮食诱导的胰岛素抵抗具有抵抗性。我们发现，MAD 2也需要保持ATP结合缺陷型IR（激酶死亡）突变体在内质网（ER）。我们推测，有丝分裂调节剂维持代谢稳态施加时空控制IR信号细胞内。为了测试这一点，我们将： AIM 1.通过MAD 2建立红外时空控制的生理功能。我们的初步数据显示，IR不能与MAD 2结合的IR 4A/4A小鼠显示延迟的IR内吞作用和延长的IR信号传导。我们推测，IR运输MAD 2控制葡萄糖和脂质代谢。我们将分析IR 4A/4A小鼠的代谢表型，使用野生型（WT）、肝脏特异性p31-/-（肝脏-p31-/-）和肝脏-IR-/-小鼠作为对照。我们还将使用小鼠和培养细胞中SHP 2的化学抑制剂测试IR时空控制在代谢中的作用。 AIM 2.确定IR-MAD 2结合促进肝脏脂肪生成的机制。mTOR复合物（mTORC）通过激活肝脏中的SREBP 1促进脂质合成11，12。mTORC 1还通过负反馈环13-17衰减IR信号传导。我们的初步数据显示，IR 4A/4A小鼠在SREBP 1激活和从头脂肪生成方面显示出肝脏缺陷，尽管PI 3 K-AKT通路（mTORC 1的重要激活剂）在肝脏中增强。我们推测，IR运输MAD 2促进肝脏脂肪生成通过mTORC-SREBP 1途径或通过负反馈回路。我们将测试内吞的IR是否仍然可以保留部分活性，并在局部发出信号以促进脂肪生成。如果是这样，那么我们将确定控制肝脏脂肪生成所需的内吞IR激酶的分子靶点。我们还将研究IR-MAD 2结合在小鼠和培养细胞中mTORC 1依赖性负反馈回路中的作用。 AIM 3.阐明了MAD 2和PTP 1B对IR的质量控制和转运机制。受体酪氨酸磷酸酶PTP 1B参与PM和ER 18-22的IR信号传导调节。我们的初步结果表明，ATP结合缺陷的IR突变体，但不是催化死IR突变体，保留在ER。引人注目的是，IR-MAD 2相互作用的破坏和PTPlB的敲除都导致从ER释放ATP结合缺陷型IR突变体。我们推测PTP 1B和MAD 2通过隔离ER中ATP结合缺陷的IR来增加PM的功能性IR水平。我们将探讨MAD 2和p31 comet是否促进ER中的IR-PTP 1B相互作用并促进保留的IR的降解。如果是这样，那么我们将确定MAD 2-PTP 1B如何控制这种降解。通过cryo-EM确定PTP 1B-IR-MAD 2-p31彗星复合物的结构，我们将阐明MAD 2-PTP 1B调控IR质量控制和运输的机制。 我们的创新方法结合小鼠遗传学，细胞生物学，生物化学，冷冻EM和基因组学研究有丝分裂调节剂在代谢稳态中的作用，将促进我们对时空IR信号的调控机制的理解。重要的是，IR信号传导和纺锤体检查点机制从小鼠到小鼠都是高度保守的，这将有助于开发用于治疗2型糖尿病的临床应用。