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Insulin receptor substrates (IRSs) are phosphorylated by IGF-I receptor tyrosine kinase in a ligand-dependent manner. In turn, they bind to and activate effector proteins such as PI3K, leading to various cell responses including cell proliferation. We had reported that ubiquitin ligase Nedd4 induces mono-ubiquitination of IRS-2, thereby enhancing IRS-2 tyrosine phosphorylation, leading to increased IGF signaling and mitogenic activity. Here we show that ubiquitin-specific protease 15 (USP15) antagonizes the effect of Nedd4 on IRS-2. We identified USP15 as a protein that preferentially bound to IRS-2 when IRS-2 was conjugated with ubiquitin. In HEK293 cells, Nedd4 overexpression induced IRS-2 ubiquitination, which was decreased by USP15 co-expression while increased by USP15 knockdown. Nedd4 overexpression enhanced IGF-I-dependent IRS-2 tyrosine phosphorylation, and USP15 co-expression suppressed it. Conversely, USP15 knockdown increased IRS-2 tyrosine phosphorylation and downstream signaling in prostate cancer PC-3 cells. We concluded that USP15 attenuates IGF-I signaling by antagonizing Nedd4-induced IRS-2 ubiquitination.

Cell-surface receptors govern the critical information passage from outside to inside the cell and hence control important cellular decisions such as survival, growth, and differentiation. These receptors, structurally grouped into different families, utilize common intracellular signaling-proteins and pathways, yet promote divergent biological consequences. In rapid processing of extracellular signals to biological outcomes, posttranslational modifications offer a repertoire of protein processing options. Protein ubiquitination was originally identified as a signal for protein degradation through the proteasome system. It is now becoming increasingly recognized that both ubiquitin and ubiquitin-like proteins, all evolved from a common ubiquitin structural superfold, are used extensively by the cell and encompass signal tags for many different cellular fates. In this chapter we examine the current understanding of the ubiquitin regulation surrounding the insulin-like growth factor and insulin signaling systems, major members of the larger family of receptor tyrosine kinases (RTKs) and key regulators of fundamental physiological and pathological states.

Insulin receptor substrates (IRSs) play central roles in insulin/insulin-like growth factor (IGF) signaling and mediate a variety of their bioactivities. IRSs are tyrosine-phosphorylated by activated insulin receptor/IGF-I receptor tyrosine kinase in response to insulin/IGF, and are recognized by signaling molecules possessing the SH2 domain such as phosphatidylinositol 3-kinase (PI3K), leading to the activation of downstream pathways. Recent studies have suggested that degradation of IRSs by the ubiquitin-proteasome pathway leads to impaired insulin/IGF signaling, but the precise mechanism underlying the process is still unclear. In this study, we identified deubiquitinating enzyme ubiquitin specific protease 7 (USP7) as an IRS-2-interacting protein and demonstrated that deubiquitinase activity of USP7 plays important roles in IRS-2 stabilization through the ubiquitin-proteasome pathway. In addition, insulin treatment dissociated USP7 from IRS-2, leading to degradation of IRS-2. This dissociation was prevented by treatment with LY294002, a PI3K inhibitor, indicating that insulin activation of the PI3K pathway leads to dissociation of IRS-2 from USP7 and IRS-2 degradation. We obtained similar results for IRS-1 in cells treated with insulin and for IRS-2 in cells treated with IGF-I. Taken together, this is the first report demonstrating that USP7 is an IRS-1/2 deubiquitinating enzyme forming a negative feedback loop in insulin/IGF signaling.

Insulin-like peptides, such as insulin-like growth factors (IGFs) and insulin, induce a variety of bioactivities, such as growth, differentiation, survival, increased anabolism, and decreased catabolism in many cell types and in vivo. In general, IGFs or insulin bind to IGF-I receptor (IGF-IR) or insulin receptor (IR), activating the receptor tyrosine kinase. Insulin receptor substrates (IRSs) are known to be major substrates of receptor kinases, mediating IGF/insulin signals to direct bioactivities. Recently, we discovered that IRSs form high-molecular-mass complexes (referred to here as IRSomes) even without IGF/insulin stimulation. These complexes contain proteins (referred to here as IRSAPs; IRS-associated proteins), which modulate tyrosine phosphorylation of IRSs by receptor kinases, control IRS stability, and determine intracellular localization of IRSs. In addition, in these complexes, we found not only proteins that are involved in RNA metabolism but also RNAs themselves. Thus, IRSAPs possibly contribute to modulation of IGF/insulin bioactivities. Since it is established that disorder of modulation of insulin-like activities causes various age-related diseases including cancer, we could propose that the IRSome is an important target for treatment of these diseases.