Amino Acid Activation of mTORC1 by a PB1-Domain-Driven Kinase Complex Cascade

Abstract

SUMMARY The mTORC1 complex is central to the cellular response to changes in nutrient availability. The signaling adapter p62 contributes to mTORC1 activation in response to amino acids, and interacts with TRAF6, which is required for the translocation of mTORC1 to the lysosome and the subsequent K63-polyubiquitination and activation of mTOR. However, the signal initiating these p62-driven processes was previously unknown. Here we show that p62 is phosphorylated via a cascade that includes MEK3/6 and p38δ and is driven by the PB1-containing kinase MEKK3. This phosphorylation results in the recruitment of TRAF6 to p62, the ubiquitination and activation of mTOR, and the regulation of autophagy and cell proliferation. Genetic inactivation of MEKK3 or p38δ mimics that of p62 in that it leads to inhibited growth of PTEN-deficient prostate organoids. Analysis of human prostate cancer samples showed upregulation of these three components of the pathway, which correlated with enhanced mTORC1 activation.

ATF4-Induced Metabolic Reprograming Is a Synthetic Vulnerability of the p62-Deficient Tumor Stroma

“…No differences in number of GFP-LC3-positive puncta (Figures S1H and S1I) or in the levels of the lipidated form of LC3, LC3-II in basal conditions or in the presence of Bafilomycin A1, an inhibitor of autophagosomal and lysosomal fusion existed between both genotypes in Gln-deprived conditions (Figure S1J). Similar results were obtained when the autophagic flux was measured using the reporter GFP-mCherry-LC3 (Linares et al, 2015), which allows the identification of autolysosomes (mCherry-positive/GFP-negative: red dots) and autophagosomes (mCherry-positive/GFP-positive; yellow dots). Consistently, no changes were observed in the total number of autophagosomes and autolysosomes in p62-deficient cells as compared to normal controls in Gln-deprived conditions (Figures S1K and S1L).…”

“…Indeed, p62 has recently emerged as an important activator of mTORC1 and of ROS detoxifying pathways (NF-κB and NRF2), which are all instrumental in the oncogenic transformation of epithelial cells (Duran et al, 2011; Linares et al, 2015; Umemura et al, 2016). Notably, p62 deficiency in prostate stromal fibroblasts results in the reduction of mTORC1 activity without changes in NF-κB or NRF2, promoting an inflammatory response and the expression of TGFβ, which are both instrumental in the activation of the CAF differentiation program (Valencia et al, 2014).…”

Metabolism shapes the tumor microenvironment

“…For instance, metabolic reprogramming of prostate CAFs is mediated by the downregulation of the signaling adaptor Sqstm1 (p62), which impairs metabolic detoxification and increases IL-6 secretion through inhibition of the mTORC1/c-Myc axis, which, in turn, promotes inflammation and tumorigenesis [20]•. Conversely, in the prostate tumor epithelium, p62-dependent mTORC1 activation in response to amino acids promotes tumor proliferation [27] (Figure 3A). A similar cell-dependent role for p62 is also found in liver tumors.…”

“…While p62 accumulation in hepatocytes, due to partial autophagy inhibition, inflammation, and oxidative stress, leads to non-mutational activation of mTORC1 that can initiate hepatocellular carcinoma (HCC) development [28]••, hepatic stellate cell (HSC)-specific KO of p62 potentiates liver fibrosis, inflammation, and HCC progression [29]••. Importantly, full-body KO of p62, in the context of a commonly used HCC inducer cocktail, diethylnitrosamine (DEN), and high-fat diet administration, enhanced HCC development [29]••, which suggests that the tumor-suppressor role of p62 in the stromal fibroblast compartment [20,23,29] outweighs its tumor-promoting effects in the tumor epithelium [27–31]. Mechanistic studies revealed that p62 loss in the HSC compartment reduced the anti-fibrotic and anti-inflammatory effects of vitamin D receptor (VDR) agonists, thus promoting HSC activation [29]•• (Figure 3B).…”

Biochemical Basis of Sestrin Physiological Activities

“…It is also worth noting that, in addition to being phosphorylated by ULK1, Sestrin2 can potentiate ULK1-dependent phosphorylation [45] and degradation [13] of SQSTM1. Considering that SQSTM1 was recently characterized as an important mTORC1 regulator that is involved in amino acid sensing [68–70], future studies are necessary to understand how Sestrin2, ULK1, SQSTM1 and GATOR2 are functionally connected with each other in the context of amino acid sensing.…”