mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide
2010; Springer Nature; Volume: 29; Issue: 23 Linguagem: Inglês
10.1038/emboj.2010.271
ISSN1460-2075
AutoresWon Jun Oh, Chang -chih Wu, Sung Jin Kim, Valeria Facchinetti, Louis -André Julien, Monica Finlan, Philippe P. Roux, Bing Su, Estela Jacinto,
Tópico(s)Ubiquitin and proteasome pathways
ResumoArticle2 November 2010free access mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide Won Jun Oh Won Jun Oh Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Search for more papers by this author Chang -chih Wu Chang -chih Wu Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Search for more papers by this author Sung Jin Kim Sung Jin Kim Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Search for more papers by this author Valeria Facchinetti Valeria Facchinetti Department of Immunology, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Louis -André Julien Louis -André Julien Department of Pathology and Cell Biology, Université de Montréal, Institute for Research in Immunology and Cancer, Montréal, Quebec, Canada Search for more papers by this author Monica Finlan Monica Finlan Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Search for more papers by this author Philippe P Roux Philippe P Roux Department of Pathology and Cell Biology, Université de Montréal, Institute for Research in Immunology and Cancer, Montréal, Quebec, Canada Search for more papers by this author Bing Su Bing Su Department of Immunobiology and Vascular Biology and Therapeutics Program, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Estela Jacinto Corresponding Author Estela Jacinto Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Search for more papers by this author Won Jun Oh Won Jun Oh Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Search for more papers by this author Chang -chih Wu Chang -chih Wu Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Search for more papers by this author Sung Jin Kim Sung Jin Kim Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Search for more papers by this author Valeria Facchinetti Valeria Facchinetti Department of Immunology, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Louis -André Julien Louis -André Julien Department of Pathology and Cell Biology, Université de Montréal, Institute for Research in Immunology and Cancer, Montréal, Quebec, Canada Search for more papers by this author Monica Finlan Monica Finlan Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Search for more papers by this author Philippe P Roux Philippe P Roux Department of Pathology and Cell Biology, Université de Montréal, Institute for Research in Immunology and Cancer, Montréal, Quebec, Canada Search for more papers by this author Bing Su Bing Su Department of Immunobiology and Vascular Biology and Therapeutics Program, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Estela Jacinto Corresponding Author Estela Jacinto Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA Search for more papers by this author Author Information Won Jun Oh1,‡, Chang -chih Wu1,‡, Sung Jin Kim1, Valeria Facchinetti2, Louis -André Julien3, Monica Finlan1, Philippe P Roux3, Bing Su4 and Estela Jacinto 1 1Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA 2Department of Immunology, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA 3Department of Pathology and Cell Biology, Université de Montréal, Institute for Research in Immunology and Cancer, Montréal, Quebec, Canada 4Department of Immunobiology and Vascular Biology and Therapeutics Program, Yale University School of Medicine, New Haven, CT, USA ‡These authors contributed equally to this work *Corresponding author. Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, 683 Hoes Lane, Piscataway, NJ 08854, USA. Tel.: +732 235 4476; Fax: +732 235 5038; E-mail: [email protected] The EMBO Journal (2010)29:3939-3951https://doi.org/10.1038/emboj.2010.271 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Coordinated control of mRNA translation and processing of nascent polypeptides is critical for normal cell function, but the mechanisms that couple these events remain unclear. Deregulated protein synthesis and processing underlie a number of pathological conditions (Macario and Conway de Macario, 2005; Sonenberg and Hinnebusch, 2009). Insights on how translation and protein processing are connected have been gained on studies of the unfolded protein response, involving secretory and transmembrane proteins, but little is known on cytosolic proteins such as protein kinases (Frydman, 2001; Young et al, 2004). The mammalian target of rapamycin (mTOR) is an atypical protein kinase that forms two structurally distinct complexes, mTORC1 and mTORC2 (Polak and Hall, 2009). The rapamycin-sensitive mTORC1 regulates translation initiation and ribosome biogenesis (Proud, 2007; Ma and Blenis, 2009; Sonenberg and Hinnebusch, 2009). mTORC1, consisting of the evolutionarily conserved mTOR, raptor, and mLST8, interacts with the translation initiation complex. In the presence of growth signals, such as nutrients and growth factors, mTORC1 phosphorylates the initiation regulators, S6K and 4E-BP, resulting in the modulation of a number of initiation factors and the assembly of the 48S initiation complex (Holz et al, 2005; Dann et al, 2007). The cellular function of mTORC2, consisting of mTOR, rictor, SIN1, and mLST8, is less clear but so far includes actin cytoskeleton reorganization and cell survival (Jacinto et al, 2004, 2006; Sarbassov et al, 2004). The mTORC2-mediated phosphorylation of the antiapoptotic proteins Akt/PKB and/or SGK could promote cell survival (Sarbassov et al, 2005; Jacinto et al, 2006; Alessi et al, 2009). mTORC2 phosphorylates the hydrophobic motif (HM) and turn motif (TM) sites of several members of the AGC (protein kinase A, PKG, PKC) kinase family, such as Akt, PKC, and SGK (Sarbassov et al, 2005; Facchinetti et al, 2008; Garcia-Martinez and Alessi, 2008; Ikenoue et al, 2008; Jacinto and Lorberg, 2008; Lee et al, 2010). Many members of this family, including the mTORC1-controlled S6K, become phosphorylated at these conserved motifs by poorly defined mechanisms (Newton, 2003; Hauge et al, 2007). The TM and HM are part of the carboxyl-terminal tail (C-tail; Figure 1A), a segment that characterizes AGC kinases and interacts with the N- and C-lobes of the kinase domain (Yang et al, 2002; Kannan et al, 2007). HM phosphorylation of Akt is induced by growth factors, necessary for full Akt activation, and often upregulated in cancer cells (Alessi et al, 1996; Sarbassov et al, 2005; Jacinto et al, 2006). In contrast, TM phosphorylation is constitutive (Alessi et al, 1996; Bellacosa et al, 1998). In the absence of TM phosphorylation, the stability of Akt and conventional PKC (cPKC) is dependent on the folding chaperone Hsp90 to prevent ubiquitination and degradation (Facchinetti et al, 2008; Ikenoue et al, 2008). Thus, TM phosphorylation is critical for the proper C-tail folding and stability of Akt and cPKC. 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. Download figure Download PowerPoint In this study, we demonstrate that constitutive phosphorylation of the TM site of Akt occurs during translation and that mTORC2 associates with actively translating ribosomes (polysomes) to fulfill this function. These findings unravel that mTORC2, like mTORC1, functions in translation and has a role in cotranslational folding of nascent cytosolic polypeptides such as Akt. Results The phosphorylation of Akt at the TM site, but not the HM site, occurs during translation We sought to determine why phosphorylation of the TM and HM sites of Akt are regulated differently, yet are both mTORC2-dependent events. As we have previously shown that mTOR mediates phosphorylation of the Akt TM site in vivo and in vitro (Facchinetti et al, 2008), we hypothesized that it phosphorylates these two sites under different cellular contexts. Although HM phosphorylation occurs post-translationally on Akt membrane localization, the constitutive nature of TM phosphorylation hints that it happens during or shortly after translation. To test if TM phosphorylation occurs during translation, we developed a coupled in vitro translation/kinase assay using translation components from bacteria, wherein no TORC orthologues exist. In the absence of added HA–mTOR as the kinase source, in vitro-translated Akt lacked TM phosphorylation (Figure 1B). Upon inclusion of HA–mTOR, an increase in TM phosphorylation (Thr450) was observed simultaneously with the appearance of total Akt at 60 min. Furthermore, TM phosphorylation and total Akt synthesis increased concurrently with prolonged in vitro translation reaction (Figure 1B). The absence of lag time between synthesis and TM phosphorylation suggests that this phosphorylation event likely occurs during translation. In contrast, HM phosphorylation was hardly observed under these conditions (Figure 1B) but was detectable at longer incubation (Supplementary Figure S1), suggesting post-translational phosphorylation. Next, we analysed if TM phosphorylation during translation can also be detected in vivo. We purified polysomes from serum-restimulated HeLa cells that were treated with cycloheximide (CHX) before harvest in order to preserve intact polysomes. As expected, most total and phosphorylated Akt are present in the cytosolic (C) fractions (Figure 1C). Upon overexposure of immunoblots, we observed the presence of Akt (Akt total) in the 40 s and 60/80 s fractions (collectively labelled as monosomes M), as well as small but detectable amounts in the polysome-containing (P) fractions. TM phosphorylation in the different fractions displayed similar patterns as total Akt and is detected in the monosome (fractions 4–6) and high-density, polysome-containing fractions (fractions 7–9) (Figure 1C). Strikingly, Akt HM phosphorylation is practically undetectable in the monosome and polysome fractions, but was highly abundant in the cytosolic fractions (fractions 1–3). As these cells were serum restimulated and that HM phosphorylation of Akt is induced under this condition, these results indicate that a minuscule fraction of Akt, most likely associated with translating ribosomes, becomes phosphorylated at the TM but not at the HM site. To confirm that the phospho-TM-containing fractions (7–9) originate from polysome complexes and not other protein complexes comigrating at high-density fractions, we treated the cell extracts with RNase to disrupt the ribosomes. The small ribosomal protein S6 and the large ribosomal protein L23 that are both abundant in polysomes in non-RNase-treated cells shifted to the cytosolic and monosome fractions upon RNase treatment, confirming the disruption of ribosomes (Figure 1C). In RNase-treated extracts, TM phosphorylation diminished significantly in the high-density fractions (5–9) but accumulated in the low-density fractions (1–4) (Figure 1C). HM phosphorylation remained in the cytosol. We further verified if TM phosphorylation occurs during translation using another cell line, murine embryonic fibrolasts (MEFs), and another drug, puromycin, to specifically disassemble polysomes in vivo. Likewise, TM, but not HM, phosphorylation is present in polysomes in these cells (Figure 1D). In puromycin-treated MEFs, TM phosphorylation was essentially absent in high-density fractions. Taken together, these results indicate that TM, but not HM, phosphorylation of Akt occurs during translation. TM phosphorylation by mTORC2 is cotranslational because the Akt TM site is accessible during synthesis of the nascent polypeptide We considered how the Akt TM site, but not the HM site, could be phosphorylated during translation. Structural studies of ribosomes from lower organisms predict that the ribosomal tunnel where nascent polypeptides traverse before exiting the ribosome can accommodate a linear polypeptide of about 30 amino acids (aa) (Nissen et al, 2000; Bhushan et al, 2010). It is believed that these last C-terminal residues are shielded in the tunnel before the newly synthesized protein is extruded from the ribosome. The TM phosphorylation site is 30 aa residues away, whereas the HM site (Ser473) is only 7 aa away from the terminal residue (Figure 1A). Hence, the TM site may be accessible for phosphorylation, whereas the HM site is only post-translationally regulated due to its inaccessibility. Therefore, we lengthened the C terminus of Akt by ligating either 15 or 30 additional aa residues, making the HM site either 22 aa (Akt–His) or 37 aa (Akt–HA–His2X) away from the final residue (Supplementary Figure S2). Using both long-tail Akt template and N-terminally tagged His–Akt, we found equivalent phosphorylation of the TM site by HA–mTOR (Figure 2A). However, HM phosphorylation became more efficient using both long-tail Akt but not N-terminally tagged His–Akt. Because the HM site of Akt–His is only 22 aa away from the terminal residue and predictably should be protected by the tunnel, we questioned whether this phosphorylation is co- or post-translational in comparison with the TM site. Using the Akt–His template, we performed in vitro translation but added HA–mTOR only after terminating the reaction by treatment with neomycin and RNase. Under this condition, TM site phosphorylation of Akt–His was undetectable (Figure 2B, lane 3), whereas HM phosphorylation diminished only slightly. These findings clearly illustrate that the TM is not post-translationally phosphorylated, whereas the HM site of Akt–His can undergo post-translational phosphorylation by mTOR. Without neomycin/RNase treatment and when the in vitro translation reaction was allowed to continue for another hour after addition of HA–mTOR (Figure 2B, lane 4), Akt continued to be translated as indicated by an increase in total Akt–His. During this additional hour of incubation in the presence of HA–mTOR, the level of TM phosphorylation did not correspond to total Akt–His levels. In fact, it was equivalent to the amount observed in lane 2, wherein HA–mTOR was included throughout the 1-h reaction. This suggests that only a fraction of total Akt (in lane 4), most likely the newly synthesized polypeptides coming from the second hour reaction, became phosphorylated at this site. In contrast, HM phosphorylation is equivalent to total Akt–His expression. This demonstrates that both the released polypeptides from the first hour of reaction and the newly synthesized polypeptides from the second hour reaction became phosphorylated at the HM upon delayed addition of mTOR. These results reveal that mTORC2 phosphorylates the TM site exclusively during translation and that it cannot phosphorylate this site after the Akt polypeptide is released from the ribosome. 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. Download figure Download PowerPoint Next, we truncated the C-tail of Akt such that the TM site is only 14 aa from the terminal residue. Using this construct, we noted that our commercial Akt (total) antibody does not recognize this truncated Akt (Figure 2C). However, when we used a T7 antibody to detect an epitope that is present on the N terminus of our His–Akt constructs (Supplementary Figure S2), the truncated His–Akt along with the full-length and intermediate forms of the other His–Akt constructs were apparent, confirming the presence of in vitro-translated products (Figure 2C and Supplementary Figure S3) (Note: We did not use anti-His antibody, as the bacterial translation components are His-tagged). In contrast to the wild-type and long-tail (His–Akt–His) Akt, we did not observe TM phosphorylation of truncated His–Akt even at prolonged incubation (Figure 2C). These results demonstrate that mTOR cannot phosphorylate the TM site when rendered inaccessible, but more importantly, the TM, unlike the HM, cannot be phosphorylated post-translationally. Alternatively, truncation of the C-tail of Akt does not allow TM phosphorylation due to conformational constraints. We then further analysed the role of mTORC2 in phosphorylating the TM site during translation. Both the TM and HM phosphorylation required the kinase activity of mTOR, as little to no phosphorylation was observed using the kinase-dead HA–mTOR (Figure 2D). The phosphorylation observed for the TM site is indeed specific to this Thr residue, as mutation to an Ala abolished phosphorylation in the presence of mTOR (Figure 2E). As we obtained inefficient phosphorylation of Akt during translation using rictor immunoprecipitates (data not shown), we used pharmacological inhibition of mTOR complexes to distinguish the kinase activity of mTORC1 versus mTORC2. Before harvest and purification of HA–mTOR from HEK293 cells, we inhibited mTORC1 by treatment with rapamycin. During the translation reaction, we added Torin1, the mTOR active site inhibitor that blocks both mTORC1 and mTORC2 (Thoreen et al, 2009). Inhibition by rapamycin did not block phosphorylation of Akt, whereas Torin1 abolished phosphorylation at both TM and HM sites (Figure 2F). Because previous studies demonstrate that mTORC2 also controls phosphorylation of cPKC at the homologous TM and HM sites (Sarbassov et al, 2004; Guertin et al, 2006; Facchinetti et al, 2008; Ikenoue et al, 2008), we examined if we can reconstitute the mTORC2-dependent phosphorylation of PKC using coupled in vitro translation/kinase assay. Using a PKCα construct with an extended tail, we observed efficient phosphorylation at both the TM and HM sites in the presence of HA–mTOR (Figure 2G). Thus, our results collectively demonstrate that mTOR, as part of an intact mTORC2, phosphorylates its target sites in Akt and PKC when these sites are accessible during translation. Akt is cotranslationally phosphorylated using in vitro and in vivo eukaryotic systems To verify if we can reconstitute cotranslational TM phosphorylation in a eukaryotic system, we used rabbit reticulocyte lysates for the in vitro translation reaction. Because there was abundant Akt in this system (Figure 3A), we used lengthened akt templates (Supplementary Figure S2) whose products migrate at higher MW in SDS–PAGE, enabling us to detect in vitro-translated Akt. mTORC2 components were also present in the reticulocyte lysates (data not shown). In vitro-translated His–Akt increased over time corresponding to enhanced TM site phosphorylation (Figure 3A). His–Akt phosphorylation, but not phosphorylation of the rabbit Akt, was blocked when Torin1 was included during the reaction, indicating that mTOR mediated the phosphorylation of in vitro-translated Akt. On the other hand, HM phosphorylation was inefficient but detectable after 2 h translation reaction (Figure 3A). When we used the long-tail template akt–ha–his2X or his–akt–his, we observed efficient phosphorylation of both the TM and HM sites, which can be diminished by Torin1 (Figure 3B and C). These results demonstrate that the mTOR-mediated cotranslational phosphorylation of Akt can be reconstituted in vitro using rabbit reticulocyte lysates. 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. Download figure Download PowerPoint We next tested how the long-tail Akt can be phosphorylated in vivo. We expressed HA–Akt or long-tail Akt in MEFs. Unlike the TM site, wherein starvation did not abolish phosphorylation, the HM site of long-tail Akt became dephosphorylated (Supplementary Figure S4). This further confirms that the HM, but not the TM, can be post-translationally regulated and that the TM phosphorylated Akt is resistant to starvation-induced dephosphorylation. Hence, to ascertain if the long-tail Akt can undergo cotranslational phosphorylation in vivo, we purified polysomes from cells expressing Akt–HA–His2X. Unlike endogenous wild-type Akt, HM phosphorylation was observed in high-density fractions (7–9) and this phosphorylation shifted to lower density fractions upon RNase treatment (Figure 3D). In contrast, there was little to no phosphorylation of the activation loop in high-density fractions. Thus, the HM site of long-tail Akt can be phosphorylated cotranslationally in vivo, but is still subject to post-translational regulation in response to growth conditions. It was reported that mTOR active site inhibitor blocked the HM, but not TM phosphorylation of Akt (Feldman et al, 2009). Our findings above suggest that as TM phosphorylation occurs during translation and is resistant to starvation-induced dephosphorylation, pre-existing Akt would remain phosphorylated. Therefore, only the pool of nascent Akt would not undergo TM phosphorylation upon mTOR inhibition. Because Akt has a half-life of about 36 h (Basso et al, 2002), we would therefore expect the appearance of non-phosphorylated Akt only after prolonged mTORC2 inhibition. Indeed, upon incubation of MEF cells with Torin1, acute treatment did not abolish TM phosphorylation (Figure 3E). Attenuation of TM phosphorylation was only evident after 24-h incubation. On the other hand, HM phosphorylation was abolished at all time points. These results suggest that on mTOR inhibition, pre-existing Akt does not become dephosphorylated at the TM but newly synthesized Akt cannot undergo TM and HM phosphorylation. Collectively, the above results confirm that the TM site undergoes cotranslational phosphorylation, whereas HM phosphorylation happens post-translationally because the HM site is not accessible during translation. mTORC2 is required for efficient translation, but is not part of the translation initiation complex As mTORC2 can phosphorylate Akt during translation and given that mTORC1 function involves translation initiation, we questioned whether mTORC2 also has a role in translation. By metabolic labelling of newly synthesized proteins, we found that although there was no difference at early time points as previously reported (Thoreen et al, 2009), a marked decrease in translation occurred by 60 min in serum-restimulated SIN1−/− cells in comparison with wild type (Figure 4A). Re-expression of SIN1β in SIN1−/− cells restored translation levels, confirming that the defect is due to the absence of SIN1. Treatment of cells using Torin1 further diminished translation at longer time point (60 min) in both wild-type and SIN1−/− MEFs, suggesting that both mTORC1 and mTORC2 can function in translation (Supplementary Figure S5). The previous findings that phosphorylation of the translation initiation regulators S6K and 4E-BP1 in mTORC2-disrupted cells is not attenuated suggested that mTORC2 may not control early initiation events (Guertin et al, 2006; Jacinto et al, 2006; Shiota et al, 2006). To verify this, we compared the binding of raptor versus rictor to the initiation complex containing translation initiation factors. Indeed, raptor, but not rictor, associates robustly with the 7-methylguanosine cap complex and also with eIF3β in 40S-containing fractions (Figures 4B and C). Hence, mTORC2 is probably not involved in early initiation signalling events. Next, when we examined eEF2 dephosphorylation, an event that enhances translation elongation (Proud, 2007), eEF2 had decreased phosphorylation at Thr56, most evident from 5 to 40 min after serum restimulation in wild-type cells (Figure 4D). In SIN1−/− MEFs, this dephosphorylation was not discernible (Figure 4D) but became evident upon SIN1 re-expression (Figure 4E). Furthermore, dephosphorylation of eEF2 in Torin1-treated wild-type cells was absent despite serum-repletion (Figure 4F). Taken together, these results support a role for mTORC2 in translation. Figure 4.mT
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