Abstract
Phosphorylation sites that are strong mTORC1 substrates are more resistant to rapamycin.
Major finding: Phosphorylation sites that are strong mTORC1 substrates are more resistant to rapamycin.
Clinical relevance: This property may underlie the weak antitumor activity of rapamycin in clinical studies.
Impact: Differential substrate quality may allow for hierarchical regulation of effectors based on signal strength.
The phosphorylation of many mTOR complex 1 (mTORC1) targets is resistant to the allosteric mTOR kinase inhibitor rapamycin, though the underlying reason remains a mystery. The failure of rapamycin to effectively inhibit phosphorylation of all mTORC1 targets is likely a contributing factor to its failure in several cancer clinical trials, which has led to the development of next-generation ATP-competitive mTOR inhibitors. By measuring the in vitro kinase activity of mTORC1 on short synthetic peptides incorporating single mTORC1 phosphorylation sites, Kang and colleagues found that the capacity of a site to be phosphorylated by mTORC1, or its substrate quality, was strongly correlated with whether it would be dephosphorylated in response to rapamycin. This observation suggests a model wherein the diminished kinase activity of rapamycin-bound mTORC1 is still sufficient to phosphorylate the best mTORC1 substrates, whereas the phosphorylation of poor mTORC1 substrates is most susceptible to inhibition by rapamycin. The authors identified determinants of substrate quality using a positional scanning peptide library screen that identified a strong preference of mTORC1 for a proline, hydrophobic, or aromatic residue at the +1 site and serine over threonine at the phosphoacceptor site. Mutation of mTORC1 substrate phosphorylation sites based on these principles was sufficient to convert rapamycin-resistant mTORC1 substrates to rapamycin-sensitive substrates, and vice versa, providing further support for the link between mTORC1 substrate quality and rapamycin resistance. Substrate quality also dictated which mTORC1 phosphorylation sites responded to nutrients, suggesting that this property is a general determinant of response to mTOR regulators. Variations in substrate quality may allow mTORC1 effector proteins to be phosphorylated in an ordered manner based on the strength of mTORC1 activity, a concept that may be applicable to other kinase cascades.
