Tracking mTORC1 Activity in the Nucleus: Distinct Regulation Revealed by Locationspecific Inhibition of Akt

Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) is a kinase complex that contains the protein kinase mTOR, the key scaffolding protein Raptor, and several other core components. It is known to sense diverse upstream signals, such as growth factors, nutrient and energy status. When activated, mTORC1 integrates these signals to critically regulate distinct cellular processes, including cell growth, metabolism, and autophagy. An intriguing question is how mTORC1 is spatially regulated to exert widespread controls on downstream effectors. Besides the well studied mTORC1 activation at the lysosome, the regulation of this complex within other subcellular compartments is less clear. Previously, we showed that growth factor induces nuclear mTORC1 activity by utilizing a genetically encoded mTORC1 activity reporter targeted to the nucleus, providing initial evidence for active mTORC1 in the nucleus. Here we probe the regulation of growth factor‐induced nuclear mTORC1 activity by combining location‐selective kinase inhibition, live‐cell imaging and cellular biochemistry. We developed Akt Substrate‐based Tandem Occupancy Peptide Sponge (Akt‐STOPS) that allows location‐specific perturbation of Akt, a critical upstream kinase, at distinct subcellular compartments. Expression of Akt‐STOPS in the nucleus largely suppressed the growth factor stimulated Akt activity without significant inhibition of cytosolic Akt activity. Using Akt‐STOPS, we showed that growth factor‐stimulated nuclear mTORC1 activity requires nuclear Akt activity. Furthermore, we showed that growth factor stimulation induces nuclear translocation of Raptor, which is sensitive to importin inhibition, and that Raptor forms a complex with mTOR in the nucleus . The nuclear translocation of Raptor specifically depends on the nuclear Akt activity, as expression of nuclear Akt‐STOPS significantly lowered the nuclear translocation of Raptor and reduced the phosphorylation of RanBP, a critical regulator of Ran GTPase. Lastly, localizing exogenous Raptor to the nucleus in Raptor knock‐down cells rescues nuclear mTORC1 activity. Taken together, these results reveal a new mode of dynamic regulation of mTORC1 by recruiting a critical scaffolding protein, distinct from the mode of lysosomal mTORC1 activation. Diversity in the regulation of mTORC1 could lead to specificity in functional controls and also provide opportunities for selective targeting.Support or Funding InformationThis work was supported by NIH grants, R35 CA197622 and R01 GM111665 to J.Z., and NS047101 to UCSD Microscopy Core.