Phosphoproteomic Analysis Identifies Grb10 as an mTORC1 Substrate That Negatively Regulates Insulin Signaling

Abstract

The evolutionarily conserved Ser-Thr kinase mTOR plays a critical role in regulating many pathophysiological processes. Functional characterization of the mTOR signaling pathways, however, has been hampered by the paucity of known substrates. We used large-scale quantitative phospho-proteomics experiments to define the signaling networks downstream of mTORC1 and mTORC2. Characterization of one mTORC1 substrate, the growth factor receptor-bound protein 10 (Grb10), showed that mTORC1-mediated phosphorylation stabilized Grb10, leading to feedback inhibition of the phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated, mitogen-activated protein kinase (ERK-MAPK) pathways. Grb10 expression is frequently downregulated in various cancers, and loss of Grb10 and loss of the well-established tumor suppressor phosphatase PTEN appear to be mutually exclusive events, suggesting that Grb10 might be a tumor suppressor regulated by mTORC1.

Quantitative global phosphoproteomics of human umbilical vein endothelial cells after activation of the Rap signaling pathway

“…The small response at the phosphoproteome level detected here is quite different from global quantitative phosphoproteomics studies that use cellular stimuli to directly regulate a particular kinase [72][73][74][75] or those studying cellular processes known to be greatly dependent on phosphorylation. 18,76 Much greater effects on phosphorylation throughout the entire signaling cascade are also observed, as expected.…”

Contrasting effects of chronic, systemic treatment with mTOR inhibitors rapamycin and metformin on adult neural progenitors in mice

“…In contrast, we observe that both pharmacological and genetic inhibition of mTORC1 evokes spontaneous pain, mechanical hypersensitivity, and increased sensory neuron excitability (Melemedjian et al 2013). Rapamycin releases the feedback inhibition of Grb10, which is an upstream regulator of MAPK pathway (Kebache et al 2007;Hsu et al 2011;Yu et al 2011) and has previously been shown to increase ERK activation in TSC2 −/− murine embryonic fibroblasts (Ghosh et al 2006).…”

mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR

“…However, the molecular mechanism underlying the integration of multiple signaling elements to produce a concerted response to insulin and IGF is largely unknown. Previous studies have demonstrated that mTOR is tightly involved in IGF-IR and InsR signaling, either as an effector or regulator of several key elements in signaling pathway [6,14,18,19]. mTORC1 and its substrate S6K1 promote IRS1 degradation, thereby negatively regulate IGF-IR and InsR signaling.…”

“…mTORC1 and its substrate S6K1 promote IRS1 degradation, thereby negatively regulate IGF-IR and InsR signaling. Recently, Grb10, an adaptor protein that negatively regulates InsR and IGF-IR, has been identified as an mTORC1 substrate that is stabilized by mTORC1 [18,19]. mTORC2 positively regulates Akt and ERK, two key elements in IGF-IR/InsR signaling.…”

“…IRS1 and IRS2 contain multiple tyrosine phosphorylation motifs that serve as docking sites for SH2 domain-containing proteins which in turn activate signaling pathways such as PI3K and MAPK/ERK cascades [17]. While mTORC1 acts as an effector downstream of IGF/insulin signaling and elicits feedback inhibition of IRS1 and stabilization of Grb10 [18,19], mTORC2 may positively regulate IGF signaling by phosphorylating IGF-II mRNA-binding protein to promote IGF-II translation [20,21]. In addition, mTORC2 disruption in mice leads to insulin resistance [22,23].…”