Bipartite binding and partial inhibition links DEPTOR and mTOR in a mutually antagonistic embrace

Abstract

The mTORC1 kinase complex regulates cell growth, proliferation, and survival. Because mis-regulation of DEPTOR, an endogenous mTORC1 inhibitor, is associated with some cancers, we reconstituted mTORC1 with DEPTOR to understand its function. We find that DEPTOR is a unique partial mTORC1 inhibitor that may have evolved to preserve feedback inhibition of PI3K. Counterintuitively, mTORC1 activated by RHEB or oncogenic mutation is much more potently inhibited by DEPTOR. Although DEPTOR partially inhibits mTORC1, mTORC1 prevents this inhibition by phosphorylating DEPTOR, a mutual antagonism that requires no exogenous factors. Structural analyses of the mTORC1/DEPTOR complex showed DEPTOR’s PDZ domain interacting with the mTOR FAT region, and the unstructured linker preceding the PDZ binding to the mTOR FRB domain. The linker and PDZ form the minimal inhibitory unit, but the N-terminal tandem DEP domains also significantly contribute to inhibition.

Searching for new mTOR kinase inhibitors: Analysis of binding sites and validation of docking protocols

“…As we mentioned, mTOR exists in a form of two complexes. In PDB, a few structures of mTORC1 complex and related proteins were deposited: 5FLC, 5H64, 5WBI (Raptor), 5WBJ (Raptor), 5WBK (Raptor), 6BCU, 6BCX, 6SB0, 6SB2, 7OWG (Raptor), 7PEA (Raptor), 7PEB (Raptor) and 7PEC (Raptor), (Anandapadamanaban et al, 2019; Aylett et al, 2016; Heimhalt et al, 2021; Wälchli et al, 2021; Yang et al, 2016; Yang et al, 2017). Among them, complexes 6BCX and 6BCU were obtained via electron microscopy with resolution: 3.00 and 3.80 Å, respectively.…”

Loop modelling provides insights into regulation of mTORC1 activity via DEPTOR dimerisation

“…This concurs well with an mTOR inhibition which is dependent on neither substrate identity nor linker phosphorylation, as the 13A DEPTOR is still capable of inhibiting mTORC1 3 . DEPTOR's DEP1-DEP2 domains, perplexingly, are missing in the FRB-bound structure 3 -it is unclear why the weak FRB-linker interaction with a Kd of ⁓500 μM 3 is preferred over the interaction between FAT and DEP1-DEP2 that covers almost 10 nm 2 of interface 8 . DEPTOR's oligomeric state is contentious too -DEP1-DEP2 has notably been characterised as monomeric in the presence of 1 mM TCEP 8 or DTT 9 , but dimeric in the absence of any reducing agents 10 .…”

“…3). It is noted that as the FRB binding is weak 3 , the linker might also swing towards the electropositive FAT region into a non-inhibitory mode (Fig. 3), which may explain why DEPTOR inhibits mTORC1 only partially 3 .…”

“…While the proposed mechanism for DEPTOR regulation still remains to be verified, it may help explain certain discrepancies between some studies. Specifically, phosphorylation of the linker's S293 and S299 has been suggested to promote CK1α-mediated DEPTOR inactivation via proteasomal degradation 4,5 , yet mTORC1 alone is sufficient to inactivate DEPTOR in vitro 3 . Mutating the linker's S286, S293 or S299 to alanine, perplexingly, impedes mTORC1's ability to phosphorylate and inactivate DEPTOR in vitro 4 .…”

Multifocal, multiphenotypic tumours arising from an MTOR mutation acquired in early embryogenesis

“…After 52 h (mTORC1) or 68 h (mTORC2), cells were harvested by centrifugation and cell pellets were frozen in liquid N 2 . mTORC1 was purified from cell pellets from 2 L Expi293F culture as described before, 31 by affinity purification on a Strep-Trap HP resin, followed by Strep-tag cleavage by TEV protease overnight on the column. The cleaved protein was further purified by anion-exchange chromatography (AEX) on a 5 mL HiTrap Q column (Cytiva), concentrated with Amicon Ultra-4 100 kDa concentrators, flash frozen in liquid N2 and stored at -80 °C.…”