Location-specific inhibition of Akt reveals regulation of mTORC1 activity in the nucleus

Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) integrates growth, nutrient and energy status cues to control cell growth and metabolism. While mTORC1 activation at the lysosome is well characterized, it is not clear how this complex is regulated at other subcellular locations. Here, we combine location-selective kinase inhibition, live-cell imaging and biochemical assays to probe the regulation of growth factor-induced mTORC1 activity in the nucleus. Using a nuclear targeted Akt Substrate-based Tandem Occupancy Peptide Sponge (Akt-STOPS) that we developed for specific inhibition of Akt, a critical upstream kinase, we show that growth factor-stimulated nuclear mTORC1 activity requires nuclear Akt activity. Further mechanistic dissection suggests that nuclear Akt activity mediates growth factor-induced nuclear translocation of Raptor, a regulatory scaffolding component in mTORC1, and localization of Raptor to the nucleus results in nuclear mTORC1 activity in the absence of growth factor stimulation. Taken together, these results reveal a mode of regulation of mTORC1 that is distinct from its lysosomal activation, which controls mTORC1 activity in the nuclear compartment.

A Highly Sensitive Fluorescent Akt Biosensor Reveals Lysosome-Selective Regulation of Lipid Second Messengers and Kinase Activity

“…As expected, in the cytosol compartment and the plasma membrane, PDGF induced a 325 ± 20% (Δ R / R 0 , n = 20) increase and a 127 ± 11% (Δ R / R 0 , n = 23) increase for Cyto-ExRai-AktAR2 (Figure b) and PM-ExRai-AktAR2 (Figures c and S3a), respectively. Moreover, Nuc-ExRai-AktAR2 (Figure d), colocalized with nuclear marker (DAPI), exhibited a 102 ± 13% (Δ R / R 0 , n = 13) increase in the excitation ratio in response to PDGF, in line with previous reports on nuclear Akt activity. ,, In addition, we observed PDGF-stimulated Akt activity at a previously underappreciated location, Golgi membranes. As seen in Figure e, Golgi-ExRai-AktAR2 colocalized with appropriate Golgi marker (Golgin-97) and responded to PDGF with a 169 ± 21% (Δ R / R 0 , n = 12) increase in the excitation ratio.…”

Class 3 phosphoinositide 3‐kinase promotes hepatic glucocorticoid receptor stability and transcriptional activity

“…Our findings of lower GR phosphorylation in the livers of Vps15‐LKO mice are also consistent with repressive functions of activated Akt signalling on GSK3β/α 38 . Furthermore, while Akt and GSK3 are most often discussed as cytosolic signal transduction cascade, both kinases and their different substrates (eg, FoxO1/3a, GR, PGC1a, p300, Mdm2, β‐catenin) also function in the nucleus 47‐51 . In addition, as soon as GC‐GR activity is induced, an inhibitory feed‐back loop for GR repression and degradation is activated to prevent its over‐activation 52 .…”

“…38 Furthermore, while Akt and GSK3 are most often discussed as cytosolic signal transduction cascade, both kinases and their different substrates (eg, FoxO1/3a, GR, PGC1a, p300, Mdm2, βcatenin) also function in the nucleus. [47][48][49][50][51] In addition, as soon as GC-GR activity is induced, an inhibitory feed-back loop for GR repression and degradation is activated to prevent its over-activation. 52 One of the E3 ligases that facilitates polyubiquitination for proteasomal degradation of GR is Mdm2.…”

Structure of autoinhibited Akt1 reveals mechanism of PIP3-mediated activation

“…While these experiments do not rule out the possibility of cytosolic Akt activity, the evidence suggests that Akt activation and most of its subsequent activity is dependent on PIP 3 or PI(3,4)P 2 . While it is eminently conceivable that substrates of Akt that exert their functions in the nucleus could be phosphorylated by Akt bound to PI(3,4)P 2 -rich endomembranes in the cytoplasm (55), it should be noted that evidence of nuclear Akt signaling has also recently been obtained (56). Further studies will undoubtedly be required to understand precisely how and under what conditions Akt is activated in the nucleus.…”