Proteome and Phosphoproteome Analysis of Brown Adipocytes Reveals That RICTOR Loss Dampens Global Insulin/AKT Signaling

We applied DDA proteomics along with DDA and PRM phosphoproteomics to study brown adipocytes lacking RICTOR, an essential subunit of mTOR complex 2, over a time series of insulin stimulation. RICTOR loss caused many protein abundance changes, including increased levels of interferon response proteins. The insulin phosphoproteome response in RICTOR-deficient cells was dampened, including known functional sites ACLY S455, VIM S39, and EIF4B S422, potentially helping to bridge the gap between the acute and chronic effects of RICTOR loss. [Display omitted] Highlights •Global and targeted phosphoproteomics in RICTOR-deficient brown adipocytes.•RICTOR loss leads to higher levels of many interferon response-associated proteins.•RICTOR loss dampens the dynamic insulin-dependent phosphoproteome response.•ACLY S455, VIM S39, and EIF4B S422 are among the most dampened phosphosites. Stimulating brown adipose tissue (BAT) activity represents a promising therapy for overcoming metabolic diseases. mTORC2 is important for regulating BAT metabolism, but its downstream targets have not been fully characterized. In this study, we apply proteomics and phosphoproteomics to investigate the downstream effectors of mTORC2 in brown adipocytes. We compare wild-type controls to isogenic cells with an induced knockout of the mTORC2 subunit RICTOR (Rictor-iKO) by stimulating each with insulin for a 30-min time course. In Rictor-iKO cells, we identify decreases to the abundance of glycolytic and de novo lipogenesis enzymes, and increases to mitochondrial proteins as well as a set of proteins known to increase upon interferon stimulation. We also observe significant differences to basal phosphorylation because of chronic RICTOR loss including decreased phosphorylation of the lipid droplet protein perilipin-1 in Rictor-iKO cells, suggesting that RICTOR could be involved with regulating basal lipolysis or droplet dynamics. Finally, we observe mild dampening of acute insulin signaling response in Rictor-iKO cells, and a subset of AKT substrates exhibiting statistically significant dependence on RICTOR.