The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) protein kinase promotes cell growth by controlling major anabolic and catabolic processes in response to a variety of environmental and intracellular stimuli, and is deregulated in aging and human diseases such as cancer and diabetes. Rapamycin, an allosteric inhibitor of mTORC1, is used clinically in organ transplantation and the treatment of certain cancers. Exactly how rapamycin perturbs mTORC1 signaling is poorly understood and it remains unknown why certain mTORC1 phosphorylation sites are sensitive to the drug whereas others are not. Here, we test the hypothesis that the inherent capacity of a phosphorylation site to serve as an mTORC1 substrate (a property we call substrate quality) is a key determinant of its sensitivity to rapamycin as well as nutrient and growth factor starvation.

mTORC1 Phosphorylation sites encode their sensitivity to physiological and pharmacological modulators of mTORC1. Substrate quality is an important determinant of how effectively mTORC1 phosphorylates its substrates in the response to both pharmacological and natural regulators ofthe kinase.

We measured the in vitro kinase activity of mTORC1 towards short synthetic peptides encompassing single mTORC1 phosphorylation sites and refined the established mTORC1 phosphorylation motif. We introduced subtle mutations into bona fide mTORC1 phosphorylation sites that we found to enhance or reduce their phosphorylation by mTORC1 in vitro and monitored the corresponding changes in the sensitivity of these sites to rapamycin treatment within cells. Finally, we assessed whether the modifications of the mTORC1 phosphorylation sites also altered their sensitivities to nutrient and growth factor starvation.

The response of an mTORC1 phosphorylation site to rapamycin treatment should depend on the balance between the activity of mTORC1 and of the protein phosphatase(s) that dephosphorylates it. We found that the in vitro kinase activity of mTORC1 toward peptides containing established phosphorylation sites strongly correlates with the resistance of the sites to rapamycin within cells. Moreover, the relative affinities of the mTOR kinase domain for the peptides also correlated with its capacity to phosphorylate them. In addition to a preference for either proline or a nonproline hydrophobic residue in the +1 position, our refinement of the mTORC1 phosphorylation motif revealed preferences for noncharged residues surrounding the phosphoacceptor site and for serine over threonine as the phosphoacceptor. Utilizing this improved understanding of the sequence motif specificity of mTORC1, we were able to manipulate mTORC1 activity toward its phosphorylation sites in vitro and alter their sensitivities to rapamycin treatment within cells. Interestingly, mTORC1 phosphorylation sites also varied in their sensitivities to nutrient and growth factor levels and manipulations in substrate quality were sufficient to alter their responses to nutrient and growth factor starvation.

Our findings suggest that the sequence composition of an mTORC1 phosphorylation site, including the presence of serine or threonine as the phosphoacceptor, is one of the key determinants of whether the site is a good or poor mTORC1 substrate within cells. Even though the phosphorylation of mTORC1 sites is subject to varied regulatory mechanisms, we propose that differences in substrate quality are one mechanism for allowing downstream effectors of mTORC1 to respond differentially to temporal and intensity changes in the levels of nutrients and growth factors as well as …