Spatiotemporal control of insulin signaling by mitotic regulators

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.