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. 2010 Dec 1;29(23):3939-51.
doi: 10.1038/emboj.2010.271. Epub 2010 Nov 2.

mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide

Won Jun Oh  1 , Chang-chih Wu, Sung Jin Kim, Valeria Facchinetti, Louis-André Julien, Monica Finlan, Philippe P Roux, Bing Su, Estela Jacinto
Affiliations

mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide

Won Jun Oh et al. EMBO J. .

Abstract

The mechanisms that couple translation and protein processing are poorly understood in higher eukaryotes. Although mammalian target of rapamycin (mTOR) complex 1 (mTORC1) controls translation initiation, the function of mTORC2 in protein synthesis remains to be defined. In this study, we find that mTORC2 can colocalize with actively translating ribosomes and can stably interact with rpL23a, a large ribosomal subunit protein present at the tunnel exit. Exclusively during translation of Akt, mTORC2 mediates phosphorylation of the nascent polypeptide at the turn motif (TM) site, Thr450, to avoid cotranslational Akt ubiquitination. Constitutive TM phosphorylation occurs because the TM site is accessible, whereas the hydrophobic motif (Ser473) site is concealed in the ribosomal tunnel. Thus, mTORC2 can function cotranslationally by phosphorylating residues in nascent chains that are critical to attain proper conformation. Our findings reveal that mTOR links protein production with quality control.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
The turn motif site of Akt is phosphorylated during translation in vitro and in vivo. (A) Sequence of the C-tail of murine Akt1. Akt has a pleckstrin homology (PH) domain at the N terminus. A C-tail (grey box) that is conserved among AGC kinases follows the catalytic domain and contains the conserved turn (TM) and hydrophobic motifs (HM), which get phosphorylated at Thr450 and Ser473, respectively. (B) Wild-type akt was used as template in a coupled in vitro translation (bacterial components) and kinase assay by incubating at the indicated times with mock (−) or HA–mTOR (+) immunoprecipitates from HEK293 cells. Aliquots of reaction were fractionated by SDS–PAGE and immunoblotted with the indicated antibodies. (C) HeLa cells were starved and restimulated with serum, then treated with cycloheximide (CHX). Cell extracts were untreated (−) or treated (+) with RNase then subjected to sucrose gradient fractionation. Absorbance (A260) (y axis) versus increasing density (x axis) of fractions was monitored (upper panels), and aliquots of each fraction were subjected to SDS–PAGE and western blotting using specific antibodies. Cytosolic (C), monosome (M; 40s-, 60s/80s containing), and polysome-containing (P) fractions are labelled. Both short (se) and long (le) exposures for phosphorylated Akt at the HM site are shown. (D) Wild-type MEFs were starved and restimulated with serum. Cells were then treated with CHX or puromycin and A260 profile of fractions was obtained. Fractions were processed as in 1C. Monosome (M)- and polysome (P)-containing fractions were run in SDS–PAGE and immunoblotted as in 1C.
Figure 2
Figure 2
mTOR phosphorylates the TM site only during translation, but it can phosphorylate the HM site either co- or post-translationally if the carboxyl-tail of Akt is lengthened. (A) N terminally tagged his–akt or long-tail Akt (akt–his and akt–HA–his2X) were used as templates for coupled in vitro translation (bacterial components) and kinase assay for 1 h in the presence of HA–mTOR immunoprecipitates purified from HEK293 cells. Amount of phosphorylated or total Akt was detected by immunoblotting. (B) akt–his was used for in vitro translation and kinase assay. Mock-transfected (lane 1) or HA–mTOR (lane 2) immunoprecipitates from HEK293 cells were added during (+) the entire 60 min of translation reaction. In lanes 3 and 4, HA–mTOR was only added after the first 60 min in vitro translation reaction in the presence (lane 3) or absence (lane 4) of 15 μg each of neomycin and RNase. In vitro translation/kinase assay reaction was allowed to continue for an additional 60 min after the addition of HA–mTOR in lanes 3 and 4. Phosphorylated and total proteins were detected by immunoblotting. (C) his–akt templates that are either truncated at the C terminus (trunc), full length (wt; His–Akt), or lengthened at the C terminus (-His; His–Akt–His) were subjected to in vitro translation and kinase assay as in 2A for 2 h. (D, E) In vitro translation and kinase assay using (D) wild-type HA–mTOR (WT) or kinase-dead (KD) HA–mTOR as kinase and Akt–His as substrate, (E) HA–mTOR as kinase, and either Akt–His wild-type (WT) or mutant Akt (T450A) as substrate was performed as in 2A. (F) His–Akt–His was subjected to in vitro translation/kinase assay using HA–mTOR purified from vehicle- or rapamycin-treated (1 μM, 60 min) cells. Where indicated, Torin1 (10 μM) was added during the translation reaction. (G) His–PKCα–His was used as the substrate for in vitro translation/kinase assay.
Figure 3
Figure 3
TM site phosphorylation can be reconstituted in vitro using eukaryotic translation system and is inhibited by Torin1 in vivo and in vitro. (A–C) In vitro translation of (A) his–akt, (B) akt–ha–his2X, or (C) his–akt–his template using rabbit reticulocyte lysate components was performed at the indicated times, in the absence (−) or presence of Torin1 (100 nM). (D) HeLa cells transiently transfected with Akt–HA–His2X expression construct was processed as in Figure 1C. The relative amount of each band to total bands (fractions 1–9) of the phosphorylated HM site from each group was quantitated and plotted (see bottom panel). (E) Growing MEFs were either harvested (−) or replenished and incubated with fresh media containing serum and either DMSO (vehicle) or Torin1 (250 nM) at the indicated times (minutes or hours). Total lysates were subjected to SDS–PAGE and immunoblotting. Results from four independent experiments were normalized to total Akt, averaged and fold induction relative to untreated (−) cells were plotted (lower panel). Error bars represent s.e.m.
Figure 4
Figure 4
mTORC2 is not part of the initiation complex, but is required for efficient translation. (A) Wild-type or SIN1−/− MEFs (left panel) or SIN1−/− MEFs transfected with either empty or SIN1β (SIN1β) expression vector (right panel) were starved of serum and amino acids, then metabolically labelled with 35S-Met/Cys in media containing serum for the indicated time (min). Data represent mean±s.d. from three independent experiments. (B) HeLa cells were normally grown (basal conditions [B]), or serum-starved (−) and restimulated with insulin (+). Cell lysates were incubated with 7-mGTP sepharose or control beads. Bound and total (whole cell lysates) proteins were analysed by immunoblotting. (C) Serum-restimulated wild-type MEFs were fractionated as in Figure 1C. Monosome (4–6) and polysome (7–9) fractions were immunoprecipitated with the indicated antibodies, and co-immunoprecipitated proteins were detected by immunoblotting. Rictor blots were overexposed relative to mTOR and raptor blots. (D) Wild-type or SIN1−/− MEFs were starved then restimulated with serum at different time points (min) and total extracts were analysed. A representative blot is shown (upper panel). Phosphorylated eEF2 levels were normalized to actin (loading), and mean±s.d. from three independent experiments was plotted (lower panel). (E) SIN1−/− MEFs were transfected with either empty vector (vec), HA-tagged SIN1α or SIN1β. Cells were harvested 2 days after transfection and total extracts were analysed. (F) Wild-type MEFs were starved then restimulated with serum containing either DMSO (vehicle) or 250 nM Torin1 at the indicated times (minutes). Total extracts were analysed as in D. Band signals of p-eEF2 T56 were quantitated by densitometry (arbitrary units) and expressed as mean±s.d. from three independent experiments.
Figure 5
Figure 5
An intact mTORC2 associates with ribosomal proteins and form stable interactions with rpL23a. (A) Wild-type, SIN1−/− MEFs, or HA–SIN1α-reconstituted SIN1−/− MEFs were starved (−) or starved then restimulated with serum (+). mTOR co-immunoprecipitated proteins and total lysate proteins were detected by immunoblotting. (B) HEK293 cells were transfected with either scrambled control, rictor, or raptor siRNA. mTOR was immunoprecipitated from total lysates and co-immunoprecipitated and total proteins were detected by immunoblotting. (C) HEK293 cells were co-transfected with HA–mTOR and each of the myc-tagged ribosomal protein constructs (rpL5, rpL23, or rpL23a). HA–mTOR immunoprecipitates were washed with buffer containing either 0.3% CHAPS, 1 or 0.1% Triton X-100. Co-immunoprecipitated myc-rpL proteins were detected by immunoblotting. Migration of ribosomal proteins in SDS–PAGE is indicated by arrowheads. (D, E) HEK293 cells were starved, restimulated, and treated with CHX (30 min) and protein crosslinker DSP (75 μg/ml) before harvest. Extracts were either untreated (−) or treated (+) with RNase (5 U, 30 min), followed by immunoprecipitation of either mock, rictor, SIN (D) or raptor (E) and detection of co-immunoprecipitated proteins.
Figure 6
Figure 6
An intact mTORC2 colocalizes with polysomes. (A) HeLa cell lysate fractions from Figure 1C were run on SDS–PAGE and immunoblotted with the indicated antibodies. The amount of each band relative to the combined total amount from fractions 1–9 was calculated and plotted (bottom panel). (B) Polysome profile of fractionated extracts from wild-type or SIN1−/− MEFs was obtained. Y axis represents A260. (C) Fractions from B were subjected to SDS–PAGE and analysed for the indicated proteins. More extracts from SIN1−/− MEFs were loaded to obtain comparable polyribosomal protein amounts between the two cell lines. Quantitation of the amount in cytosolic fractions (1–3) was expressed relative to the strongest band in wild-type fraction (assigned a value of 1.0), whereas amount in high-density fractions (4–9) was expressed relative to fraction 4 of wild type (assigned a value of 1.0).
Figure 7
Figure 7
Akt and PKCα become ubiquitinated during translation in the absence of mTORC2 or turn motif phosphorylation. (A) MEF cells were starved, treated with MG132, serum-restimulated and CHX-treated before harvest. Fractions obtained as in Figure 6C were immunoprecipitated using either Akt or PKCα antibodies, followed by SDS–PAGE and immunoblotting (C (fractions 1–3), M (4–6), P (7–9)). (B) HEK293 cells transiently transfected with either HA-Akt or HA-Akt–T450A were treated as in A, then fractionated, immunoprecipitated with HA antibody, followed by SDS–PAGE and immunoblotting. (C) Model for mTORC2 function during translation. mTORC2 phosphorylates the TM site (Thr450) of Akt during translation by association of mTORC2 with translating ribosomes at the vicinity of the tunnel exit. Newly released Akt polypeptide becomes fully mature and active upon phosphorylation by PDK1 at the activation loop (Thr308) and by mTORC2 at the HM (Ser473). In the absence of an intact mTORC2, total translation is defective, polysome recovery is decreased, and Akt tends to be ubiquitinated during translation.
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