Mitotic Checkpoint Regulators in Insulin Signaling

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Project Summary Insulin signaling is critical for multiple facets of animal physiology. Its dysregulation causes insulin resistance syndromes, such as type 2 diabetes. The spindle checkpoint ensures the fidelity of chromosome segregation and guards against aneuploidy. The key spindle checkpoint proteins Mad2 and BubR1 can simultaneously bind to Cdc20, converting it from an anaphase promoting complex/cyclosome (APC/C) activator to a subunit of an APC/C-inhibitory complex called the mitotic checkpoint complex (MCC). During checkpoint inactivation, a critical inhibitor of Mad2, p31comet promotes checkpoint inactivation and timely chromosome segregation. Recently, combining approaches in mouse genetics, cell biology, biochemistry, and single-cell genomics, we have discovered a critical role of the p31comet–Mad2–BubR1 module of mitotic regulators in insulin signaling through regulating insulin receptor (IR) endocytosis. In the mouse, p31comet ablation diminishes IR at the plasma membrane prior to insulin binding and causes defective insulin signaling in multiple tissues and metabolic syndrome. Mechanistically, Mad2 directly binds to IR through a canonical Mad2-interacting motif (MIM). IR-bound Mad2 facilitates BubR1-dependent recruitment of the clathrin adaptor AP2 to IR. p31comet blocks Mad2-BubR1 association and prevents spontaneous IR endocytosis. Mad2 and BubR1 are also required for insulin-stimulated IR endocytosis. This unexpected link between mitotic regulators and insulin signaling raises several outstanding questions that we wish to address in this proposal. In Aim 1, we will further elucidate the mechanism and regulation of insulin-stimulated IR endocytosis. In particular, we will determine how the newly discovered Mad2–BubR1 mechanism cooperates with previously described mechanisms to mediate proper IR endocytosis. We will establish how these mechanisms are regulated by insulin signaling. In Aim 2, we will test the intriguing hypothesis that insulin signaling reciprocally regulates the spindle checkpoint. In preliminary results, we have created a knock-in mouse (Insr4A/4A) with mutated IR alleles (4A) deficient for Mad2 binding. IR 4A cells have a weakened spindle checkpoint. We will determine the mechanisms by which IR promotes spindle checkpoint signaling through cellular and in vitro reconstitution experiments. In Aim 3, we will define the physiological functions of the mutual regulation between IR and mitotic regulators by examining the phenotypes of the Insr4A/4A mouse. We will test whether defective IR plasma membrane localization contributes to type 2 diabetes by comparing IR localization in liver biopsies from non-diabetic and diabetic patients. Collectively, the proposed research will further clarify the mechanism and function of the unexpected link between mitotic regulators and insulin signaling, and may establish the Mad2– BubR1–AP2 module as a novel therapeutic target for treating diabetes.