Depletion of Ric-8B leads to reduced mTORC2 activity

Abstract

<jats:title>Abstract</jats:title><jats:p>mTOR, a serine/threonine protein kinase that is involved in a series of critical cellular processes, can be found in two functionally distinct complexes, mTORC1 and mTORC2. In contrast to mTORC1, little is known about the mechanisms that regulate mTORC2. Here we show that mTORC2 activity is reduced in mice with a hypomorphic mutation of the Ric-8B gene. Ric-8B is a highly conserved protein that acts as a non-canonical guanine nucleotide exchange factor (GEF) for heterotrimeric Gαs/olf type subunits. We found that Ric-8B hypomorph embryos are smaller than their wild type littermates, fail to close the neural tube in the cephalic region and die during mid-embryogenesis. Comparative transcriptome analysis revealed that signaling pathways involving GPCRs and G proteins are dysregulated in the Ric-8B mutant embryos. Interestingly, this analysis also revealed an unexpected impairment of the mTOR signaling pathway.</jats:p><jats:p>Phosphorylation of Akt at Ser 473 is downregulated in the Ric-8B mutant embryos, indicating a decreased activity of mTORC2. In contrast, phosphorylation of S6, a downstream target of mTORC1, is unaltered. Knockdown of the endogenous Ric-8B gene in HEK293T cells leads to reduced phosphorylation levels of Akt at Ser 473, but not of S6, further supporting the selective involvement of Ric-8B in mTORC2 activity. Our results reveal a crucial role for Ric-8B in development and provide novel insights into the signals that regulate mTORC2 activity.</jats:p><jats:sec><jats:title>Author Summary</jats:title><jats:p>Gene inactivation in mice can be used to identify genes that are involved in important biological processes and that may contribute to disease. By using this approach, we found that the Ric-8B gene is essential for embryogenesis and for the normal development of the nervous system. Ric-8B mutant mouse embryos are smaller than their wild type littermates and show neural tube defects at the cranial region. This approach also allowed us to identify the biological pathways that are involved in the observed phenotypes, the G protein and mTORC2 signaling pathways. mTORC2 plays particular important roles also in the adult brain, and has been implicated in neurological disorders. Ric-8B is highly conserved in mammals, including humans. Our mutant mice provide a model to study the complex molecular and cellular processes underlying the interplay between Ric-8B and mTORC2 in neuronal function.</jats:p></jats:sec>

Elp3‐mediated codon‐dependent translation promotes mTORC2 activation and regulates macrophage polarization

“…As Ctu2, a tRNA-modifying enzyme acting in the same cascade as Elp3, also promotes mTORC2 activation, it is likely that these enzymes are required for the translation of mRNA candidates acting as activators of mTORC2. In this context, we identified Ric8b, a candidate known to promote mTORC2 activation (Nagai et al, 2020), as a direct target of Elp3. Ric8b expression is induced by IL-4 in macrophages, similarly to Elp3.…”

“…As Elp3 and mTORC2 deficiencies share overlapping phenotypes (Hallowell et al, 2017), we next looked for any positive regulator of mTORC2 activation whose translation may require Elp3. In this context, we got interested in Ric8b, a noncanonical guanine nucleotide exchange factor (GEF) whose deficiency impairs Akt phosphorylation on Serine 473 (Nagai et al, 2020)…”

“…Mechanistically, Elp3 deficiency impairs mTORC2 activation. Ric8b, a noncanonical guanine nucleotide exchange factor (GEF) acting as a positive regulator of mTORC2 (Nagai et al, 2020), relies on Elp3 to be properly translated in a codon-dependent manner. Both transcriptomic signature and metabolic reprogramming triggered by IL-4 is defective upon Elp3 deficiency.…”

Gα13 loss promotes tumor progression in the KPC transgenic mouse model of advanced pancreatic cancer

“…In agreement with the human data, we also found that Gα13 loss in the KPC mouse model enhanced mTOR signaling. Notably, GPCRs and G proteins have been shown to modulate mTOR signaling (Gan et al, 2019; Jewell et al, 2019; Nagai et al, 2020; Puertollano, 2019). For example, Gαs-coupled GPCRs activate protein kinase A to inhibit mTOR complex 1 in multiple cell lines and mouse tissue (Jewell et al, 2019).…”