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.
Highlights
Kinases superfamily of protein kinases, PIKKs), RAPTOR and mLST8 (Brown et al, 1994; Hara et al, 2002; Kim et al, 2003; Sabatini et al, 1994; Sabers et al, 1995). mechanistic target of rapamycin complex 1 (mTORC1) senses and integrates inputs originating from nutrients, growth factor signalling pathways, oxidative stress, and cellular energy levels (Jewell et al, 2013; Laplante and Sabatini, 2012)
Using immunoblotting with antibodies specific for the phosphorylated substrates, we found that DEPTOR inhibited mTORC1 with half maximal inhibitory concentration (IC50) of 14 μM 6 for wild-type 4E binding protein 1 (4EBP1), and 51 μM for S6K1367-404 (Figure 1A)
Several previous reports have suggested that DEPTOR dampens mTORC1 activation, rather than fully inhibiting it, leaving a residual mTORC1 specific phosphorylation of S6 kinase 1 (S6K1) (Caron et al, 2016; Dong et al, 2017; Hu et al, 2017; Laplante et al, 2012; Li et al, 2014)
Summary
Kinases superfamily of protein kinases, PIKKs), RAPTOR and mLST8 (Brown et al, 1994; Hara et al, 2002; Kim et al, 2003; Sabatini et al, 1994; Sabers et al, 1995). mTORC1 senses and integrates inputs originating from nutrients, growth factor signalling pathways, oxidative stress, and cellular energy levels (Jewell et al, 2013; Laplante and Sabatini, 2012). The effect of mTORC1 on protein synthesis is primarily due to its phosphorylation of S6 kinase 1 (S6K1) and eIF-4E binding protein 1 (4EBP1) (Gingras et al., 1999; Holz et al, 2005; Holz and Blenis, 2005; Kang et al, 2013) Both substrates interact with the mTORC1 complex through their general TOR signalling (TOS) motif that binds the RAPTOR subunit (Schalm and Blenis, 2002; Schalm et al, 2003; Schalm et al, 2005), as well as by forming a second interaction with the FRB helical insertion within the N-lobe of the mTOR kinase domain (Yang et al, 2017).
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