Spatiotemporal control of insulin signaling by mitotic regulators

具体目的：胰岛素信号通路的精确调控对动物生理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，如糖尿病5。由于2型糖尿病影响着全球超过4亿人5，了解影响该疾病的信号通路至关重要。胰岛素受体(IR)基因突变导致罕见而严重的胰岛素抵抗6。然而，2型糖尿病胰岛素抵抗的原因很多，其机制是多因素的，留下了许多悬而未决的问题。 胰岛素与质膜结合后，IR激活了分叉的信号通路：PI3K-AKT代谢通路和MAPK生长通路。活性胰岛素抵抗随后通过网状蛋白介导的内吞作用内化7。胰岛素抵抗内吞作用已被广泛研究了数十年。然而，细胞表面的功能性胰岛素抵抗在基础状态和胰岛素刺激状态下是如何被调节的，红外线转运如何影响体内特定信号通路的激活，以及红外线转运的失调如何导致人类胰岛素抵抗，目前仍不清楚。回答这些问题需要确定IR内吞作用的特定介质和调节器，并在动物模型中从机制上了解IR内吞作用的途径。 我们的长期目标是了解系统性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，MAD2，一个关键的有丝分裂调节因子，与IR底物(IRS)合作，通过将笼状蛋白适配器复合体AP2招募到IR 8-10来促进IR的内吞作用。从机制上讲，MAD2通过一个保守的MAD2相互作用基序(MIM)与IR结构性结合。MAD2抑制剂p31comet可阻断MAD2依赖的AP2向IR的募集。由MAPK和酪氨酸磷酸酶SHP2控制的IRS的磷酸化开关确保了胰岛素激活的IR的选择性内化。靶向这种反馈调节可以延长IR信号的代谢分支，并改善小鼠的胰岛素敏感性。我们的初步数据显示，IR4A/4A小鼠(缺乏MAD2结合和内吞作用)对饮食诱导的胰岛素抵抗具有抵抗力。我们发现，MAD2也是维持内质网(ER)中的ATP结合缺陷IR(KK DEAD)突变体所必需的。我们假设，有丝分裂调节剂通过对细胞内IR信号施加时空控制来维持代谢平衡。为了测试这一点，我们将： 目的1.建立MAD2对IR时空调控的生理功能。我们的初步数据显示，IR不能与MAD2结合的IR4A/4A小鼠表现出延迟的IR内吞作用和延长的IR信号。我们假设MAD2的IR转运控制着糖和脂代谢。我们将分析IR4A/4A小鼠的代谢表型，以野生型(WT)、肝脏特异性p31-/-(肝脏-p31-/-)和肝脏IR-/-小鼠为对照。我们还将使用SHP2的化学抑制剂在小鼠和培养细胞中测试IR时空调控在新陈代谢中的作用。 目的2.确定IR-MAD2结合促进肝脏脂肪生成的机制。MTOR复合体(MTORC)通过激活肝脏11、12中的SREBP1促进脂质合成。mTORC1还通过负反馈环13-17减弱IR信号。我们的初步数据显示，IR4A/4A小鼠在SREBP1激活和从头脂肪生成方面存在肝脏缺陷，尽管肝脏中的PI3K-AKT途径(mTORC1的重要激活剂)增强。我们假设MAD2的IR转运通过mTORC-SREBP1途径或通过负反馈环促进肝脏脂肪生成。我们将测试内吞的IR是否仍能保持部分活性，并在局部发出促进脂肪生成的信号。如果是这样的话，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-MAD2结合在小鼠和培养细胞中依赖mTORC1的负反馈环中的作用。 目的3.阐明MAD2和PTP1B对IR的质量控制和转运机制。受体酪氨酸磷酸酶PTP1B参与了PM和ER 18-22中IR信号的调节。我们的初步结果表明，在内质网中保留了一个ATP结合缺陷的IR突变体，而不是催化死亡的IR突变体。值得注意的是，IR-MAD2相互作用的破坏和PTP1B的敲除都导致从ER中释放出ATP结合缺陷的IR突变体。我们假设PTP1B和MAD2通过将ATP结合缺陷的IR隔离在内质网来增加PM的功能性IR水平。我们将探讨MAD2和p31comet是否促进IR-PTP1B在内质网中的相互作用，并促进保留的IR的降解。如果是，那么我们将确定MAD2-PTP1B如何控制这种降解。通过低温电子显微镜测定PTP1B-IR-MAD2-p31彗星复合体的结构，我们将阐明MAD2-PTP1B调控IR质量控制和转运的机制。 我们的创新方法结合了小鼠遗传学、细胞生物学、生物化学、冷冻-EM和基因组学来研究有丝分裂调节因子在代谢稳态中的作用，将促进我们对调控时空IR信号的机制(S)的理解。重要的是，IR信号和纺锤体检查点机制从小鼠到HU都是高度保守的，LTS将有助于开发治疗2型糖尿病的临床应用。

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.