Depletion of Ric-8B leads to reduced mTORC2 activity

Abstract

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 Gs/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.\n\nPhosphorylation 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.\n\nAuthor SummaryGene 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.