The tumor suppressor CYLD acts as a deubiquitinase for mTOR to constrain its activity.

Proper control of mTOR (mechanistic/mammalian target of rapamycin) signaling is relevant for health, disease and ageing. Information from intra- and extra-cellular signaling cues is transmitted to mTOR through an intricate signaling network that impinges on the Rag and Rheb GTPases to regulate its localization and activity. Interestingly, although mTOR is a heavily ubiquitinated protein, the role of this post-translational modification (PTM) in regulating its activation status remains poorly understood. Here, through an unbiased RNAi screen, we identified the tumor suppressor CYLD deubiquitinase (DUB) as a direct negative regulator of both mTORC1 and mTORC2 activities. Mechanistically, CYLD interacts with mTOR and removes non-degradative, K63-linked ubiquitin (Ub) chains from multiple of its residues. Consequently, CYLD loss-of-function cells are characterized by mTORC1/2 hyperactivation, elevated rates of protein synthesis, increased cell size, and resistance to serum-starvation-induced activation of cell death pathways. Moreover, silencing of