Dynamic modelling of the PI3K/MTOR signalling network uncovers biphasic dependence of mTORC1 activity on the mTORC2 subunit SIN1

The PI3K/MTOR signalling network regulates a broad array of critical cellular processes, including cell growth, metabolism and autophagy. The mechanistic target of rapamycin (MTOR) kinase functions as a core catalytic subunit in two physically and functionally distinct complexes mTORC1 and mTORC2, which also share other common components including MLST8 (also known as G beta L) and DEPTOR. Despite intensive research, how mTORC1 and 2 assembly and activity are coordinated, and how they are functionally linked remain to be fully characterized. This is due in part to the complex network wiring, featuring multiple feedback loops and intricate post-translational modifications. Here, we integrate predictive network modelling, in vitro experiments and -omics data analysis to elucidate the emergent dynamic behaviour of the PI3K/MTOR network. We construct new mechanistic models that encapsulate critical mechanistic details, including mTORC1/2 coordination by MLST8 (de)ubiquitination and the Akt-to-mTORC2 positive feedback loop. Model simulations validated by experimental studies revealed a previously unknown biphasic, threshold-gated dependence of mTORC1 activity on the key mTORC2 subunit SIN1, which is robust against cell-to-cell variation in protein expression. In addition, our integrative analysis demonstrates that ubiquitination of MLST8, which is reversed by OTUD7B, is regulated by IRS1/2. Our results further support the essential role of MLST8 in enabling both mTORC1 and 2's activity and suggest MLST8 as a viable therapeutic target in breast cancer. Overall, our study reports a new mechanistic model of PI3K/MTOR signalling incorporating MLST8-mediated mTORC1/2 formation and unveils a novel regulatory linkage between mTORC1 and mTORC2. Author summarySignalling networks are the key information-processing machineries that underpin the ability of living cells to respond proportionately to extra- (and intra-) cellular cues. The PI3K/MTOR signalling network is one of the most important signalling networks in human cells that regulates cellular response to critical hormones such as insulin; yet our understanding of the network behaviour remains far from complete. Here, we employed a highly integrative approach that combines predictive mathematical modelling, biological experimentation, and data analysis to gain novel systems-level insights into PI3K/MTOR signalling. We constructed new mathematical models of this complex network incorporating important regulatory mechanisms. In contrary to commonly-held views that mTORC2 lies upstream and is a positive regulator of mTORC1, we found that their relationship is highly non-linear and dose dependent. This finding has major implications for anti-mTORC2 therapy as depending on the cellular contexts, inhibiting mTORC2 may either reduce or enhance mTORC1 activation, the latter could inadvertently dampen the effect of mTORC2 blockade. Furthermore, our results demonstrate that MLST8 is required for the assembly and activity of both MTOR complexes and suggest MLST8 is a viable therapeutic target in breast cancer.