mTORC1 induces eukaryotic translation initiation factor 4E interaction with TOS-S6 kinase 1 and its activation

  1. Sheikh Tahir Majeed
  2. Asiya Batool
  3. Rabiya Majeed
  4. Nadiem Nazir Bhat
  5. Khurshid Iqbal Andrabi

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Abstract

Eukaryotic translation initiation factor 4E was recently shown to be a substrate of mTORC1, suggesting it may be a mediator of mTORC1 signaling. Here, we present evidence that eIF4E phosphorylated at S209 interacts with TOS motif of S6 Kinase1 (S6K1). We also show that this interaction is sufficient to overcome rapamycin sensitivity and mTORC1 dependence of S6K1. Furthermore, we show that eIF4E-TOS interaction relieves S6K1 from auto-inhibition due to carboxy terminal domain (CTD) and primes it for hydrophobic motif (HM) phosphorylation and activation in mTORC1 independent manner. We conclude that the role of mTORC1 is restricted to engaging eIF4E with S6K1-TOS motif to influence its state of HM phosphorylation and inducing its activation.

Highlights

  • Phosphorylated eIF4E interacts with TOS motif of S6 Kinase1

  • eIF4E-TOS interaction relieves S6 Kinase 1 from carboxy terminal domain auto-inhibition and primes it for activation.

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  1. eLife

    ##Author Response

    ###Reviewer #1

    1. In many instances inappropriate controls were used. For instance, a straightforward experiment to corroborate the authors model would be to employ cells that exclusively express non-phosphorylatable eIF4E mutant (such as eIF4E KI MEFs described in Furic et al., 2010) and/or MNK KOs to establish the requirement of eIF4E phosphorylation and potential cross-talk with MNK dependent mechanisms, respectively. Although there were some attempts to do this (e.g. MNK1 KD, using pharmacological inhibitors that are by the way quite non-specific), the data are insufficient to support the authors' claims. Moreover, the interaction between eIF4E and eIF4G and potential changes in the eIF4F levels that are likely to confound authors' conclusions were not assessed.
    1. Several mechanisms involving indirect effects of mTOR on eIF4E phosphorylation that have been reported in the literature were not considered. For instance, it is plausible that mTOR affects eIF4E phosphorylation by bolstering eIF4E:eIF4G association and recruitment of MNKs.

    Appropriateness of the controls to be employed is imperative. We would appreciate if controls that appear inappropriate were identified for us to improve upon. We also endorse that pharmacological inhibitors like MNK inhibitor tend to be promiscuous. However, their use in combination with knockdown experiments offers a reasonable choice for strengthening a data point. We are surprised at the insistence of the reviewer for his emphasis on indirect regulation of eIF4E phosphorylation via eIF4G and eIF4F to proximate mTORC1 and MNK response, despite the evidence herein that identifies direct regulation of this phosphorylation by mTORC1 coupled with rapamycin induced feed back response by MNK. Data generated by us so over the years including some interesting unpublished observations (Majeed R and Andrabi KI) have strengthened our contention that eIF4E phosphorylation is regulated by mTORC1 directly with eIF4E: eIF4G regulation as a back up.

    1. The evidence for direct phosphorylation of eIF4E by mTOR was based on non-optimally designed experiments. The description of methodology for the in vitro kinase assays was inadequate, and the experiment was carried out solely using GST-WTeIF4E as a substrate without appropriate controls. There also appears to be rapamycin dependent eIF4E phosphorylation in KD mTOR lanes.

    The in vitro kinase assay for eIF4E as a mTORC1 substrate has been described in detail by us previously (Batool et-al 2020). The experiment referred to, by the reviewer has been included as part of supplementary data only to serve as a ready reference.

    1. The authors use non-transformed cells as a control for eIF4E overexpression, whereby eIF4E overexpression is well-established to transform immortalized cells (Work from Sonenberg's, Bitterman's etc. labs).

    The primary data to appreciate the dynamics of eIF4E expression is represented by human tumour samples (Fig 1A-D), that clearly indicated tumour specific over-expression and eIF4E hyper-phosphorylation. In an attempt to substantiate the universality of this observation, we examined its expression across several cell lines including the ones that are not transformed. In addition, non-transformed cells were used to assess whether phosphorylation of eIF4E was a function of its over-expression which otherwise not be possible to appreciate in a tumour cell scenario.

    1. Functional assays are warranted to establish the effects of proposed mechanism on cell functions/fate.

    We appreciate the significance of functional assays and intend to include them wherever necessary.

    1. Many blots throughout the paper were of insufficient quality to be clearly interpreted.

    We would like to know which blots the reviewer is referring to.

    1. Many interpretations of the results were not justified by the data (e.g. in Figure 1C it is claimed that phosphorylation of eIF4E is increased in overexpressors, but this could be simply due to the increase in total protein levels).

    We do not believe that the enhanced phosphorylation of eIF4E is due to the increase in the total protein. As seen in Fig.1C the levels of the protein are the same throughout.

    1. Most of the work relies on transient (except for FLAG-S6K1) overexpression strategies which are prone to artifacts and not likely to represent physiological stoichiometry of investigated proteins.

    We have already used five stable cell lines. It is not possible to generate stable cells for every protein as we are studying signalling cross-talks. We believe that we have used enough positive and negative controls to rule out the possibility of artefacts.

    1. It has been previously shown (e.g. Lowe & Pelletier's labs) that eIF4E confers resistance to rapamycin by mechanisms that were clearly distinct and at least in my opinion far better substantiated than those published previously by the authors and proposed here. Indeed, eIF4E overexpression results in increased eIF4F levels, which has been shown to attenuate efficacy of not just rapamycin, but also active mTOR inhibitors, and many other oncogenic-kinase inhibitors.

    Our study although being in concert with other evidences suggesting the feedback activation of Mnk/4E pathway upon mTORC1 inhibition differs from some of the studies as quoted by the reviewer. The basic difference for this anomaly lies in the difference of the experimental conditions that we use to monitor the phosphorylation status of eIF4E, that lies from a range of 20 min to 48 hrs at 50nM concentration of Rapamycin. Studies carried out elsewhere use either 250nM conc. of rapamycin for 2hrs (Michael C. Brown-2017), 100nM for 2 hrs (Rebecca L Stead-2013) or use of rapalogs for 12 hrs (Pierre E Joubert-2015). Although, these and many other studies have implicated crosstalk to explain increase in 4E phosphorylation upon mTOR inhibition, yet they grossly fall short of comprehensively monitoring the status of 4E phosphorylation from 20 min to 2 hrs at lower conc. of rapamycin. We believe that use of higher concentration of Rapamycin allows the Mnk1 induced phosphorylation to resurface early (>3 hrs) to reconcile with the literature about the rapamycin dependent upsurge in 4E phosphorylation.

    1. Many published articles are misinterpreted as supporting the authors' claims. For instance, the authors write that "the inconsistent stature of mTORC1 as a 4EBP1 kinase in vivo" and the reference provided suggests that GSK3beta may phosphorylate 4E-BP1 in addition to mTOR which in certain contexts may lead to rapamycin resistance. As far as I understand, this, and other similar studies, do not challenge the status of mTORC1 as a 4E-BP1 kinase in vivo, but that GSK3beta (and other kinases such as Pim kinases, CDK1) may also phosphorylate 4E-BPs in certain contexts. Moreover, as initial studies on active-site mTOR inhibitors by Thoreen et al., and Feldman et al., as well as studies from Blenis' and Sonenberg's groups indicated, rapamycin does not efficiently inhibit 4E-BPs n the vast majority of contexts, which suggest that GSK3beta-dependent resistance to rapamycin may result from mTOR effectors other than 4E-BPs

    We have previously summarized the studies that question the stature of 4E-BP1 as an mTOR substrate. We would like the reviewer to go through that manuscript (Batool et al, EJCB, 2017). We have missed to cite that paper in this manuscript.

    ###Reviewer #2

    1. A large portion of Figures 1-3 is a reproduction of data from the authors' 2020 paper (Batool et al., 2020) which showed that elF4E is phosphorylated by MNK1, and that MNK1 is repressed by activation of mTORC1 signaling. While some new experiments have been added (e.g. the analysis showing increased expression of S6k1 in cancer cell lines/tissue and the in silico peptide docking analysis), these are minimal additions to the recently published work from this group.

    This study was built on our previous publication that suggest eIF4E as an important effector of mTORC1. This study however, focusses on the regulation of S6K1 and following are the additions in the paper:

    • Overexpression of eIF4E WT and S209E correlates with S6K1 phosphorylation and activity and is rapamycin-insensitive (Figure 1E, F and Supplementary Figure S1).

    • S6K1 TOS, but not HM phosphorylation is required for its interaction with eIF4E (Figure 4A, D).

    • mTORC1 is required for priming S6K1 for activation while as mTORC2 activity is responsible for phosphorylation of TOS- and CT-deficient S6K1 (Figure 5D, F).

    • Identification of a region in S6K1 that mediates mTORC2 response (Fig 6).

    • Identification of a short peptide in S6K1, which appears to interact with PHLPP1 (Fig 7).

    1. One new finding in this paper is that elF4E binds the TOS motif on S6K1 and this binding promotes the hydrophobic motif phosphorylation of S6K1. The authors interpret their data to mean that binding of elF4E induces a conformational change to relieve autoinhibition. Is there any structural information to support this conformational change? What if the binding of elF4E recruits the hydrophobic motif kinase (mTORC2 proposed) in the absence of a conformational change? There are multiple other explanations that need to be considered and addressed.

    TOS deletion/ mutation renders S6K1, inactive due to:

    The failure of hydrophobic motif (HM) to get phosphorylated implying that TOS may recruit a kinase to phosphorylate HM and activate the enzyme (prevailing model). If this were true, then phospho-mimicking HM should rescue the loss of enzyme activity due to TOS- mutation, which however is not the case.

    Or

    The failure of carboxy terminal domain (CTD) to disinhibit, implying that TOS-engagement must somehow orchestrate CTD disinhibition (conformational change) to allow HM phosphorylation as a consequence. Since loss of function due to TOS-mutation/deletion can be rescued only by CTD truncation, it is reasonable to infer that TOS engagement with 4E must serve to remove inhibition due to CTD by a change in conformation to facilitate HM phosphorylation to occur in TOS independent manner.

    Although there is no structural data, the inferences are compelling to propose the conformational change at the behest of eIF4E interaction with S6K1.

    The possibility of mTORC2 recruitment by eIF4E is not supported by any data. This is because TOS &CTD deleted variant of S6K1 continues to be phosphorylated in a torin sensitive manner (Fig 5D).

    Other consideration have also been discussed to the best of our ability.

    1. The authors propose that PHLPP1 is constitutively bound to S6K1 to suppress hydrophobic motif phosphorylation, and serum stimulation causes the release of PHLPP1 to fully activate S6K1. Unfortunately, this potentially important mechanism is experimentally addressed by only 3 co-IPs in Figure 7: overexpressed PHLPP1 co-IPs with a GST fusion with residues 78-85 of S6K1, PHLPP1 co-IPs with S6K1 (and less efficiently in the presence of serum), the PHLPP1 regulation of S6K1 is abolished in a construct in which residues 78-95 are deleted. The identification of a PHLPP1-binding determinant on S6K1 is significant but the current data just scratch the surface. What are the residues? Are they evolutionarily conserved? Are they conserved in other PHLPP1 substrates? Does the GST fusion with these 8 amino acids result in the activation of S6K1 by sequestering PHLPP1? A compelling mechanistic analysis is missing and should be provided especially since PHLPP1 is in the title of the paper.

    While deletion of sequence between 78-85 renders S6K1 non-responsive to serum stimulation, it does not affect its sensitivity towards rapamycin. Also, GST fusion of these 8 amino acids resulted in the activation of S6K1 as it sequestered PHLPP1. Some more experiments can be added to further support the contention. Three out of eight amino acids appear to be evolutionary conserved. We have performed a detailed mutagenesis of the region and the data is part of a manuscript in preparation.

    1. Deletion of residues 91- 109 inactivates S6K1, which the authors interpret as meaning the regions is critical for mTORC2 binding and HM phosphorylation. But this encompasses the Gly-rich loop and its deletion will inactivate any kinase.

    The deletion, 91-109, referred to by the reviewer, was introduced to evaluate the ability of this S6K1 variant to act as a substrate for mTORC2 mediated HM phosphorylation rather than to determine the state of S6K1 enzyme activity as perceived by the reviewer. Regardless of the influence this deletion may have in the activity state of S6K1, it should have no bearing on the ability of mTORC2 to phosphorylate S6K1at its HM situated 300 amino acids carboxy terminus to the deletion. Since this deletion results in the failure of mTORC2 to phosphorylate S6K1 at Hm, we drew following conclusion.

    • This region appeared sufficient to mediate HM phosphorylation irrespective of the presence of TOS motif.

    • That this region may support mTORC2 docking.

    • That mTORC2 mediated S6K1 phosphorylation is specific and not a random event (Refer to discussion).

    ###Reviewer #3

    1. While the authors claim that MNK1 is not the "primary" kinase phosphorylating eIF4E, they fail to show the lack of CGP57380 effect on p-eIF4E(S209) and pS6K1(T412) phosphorylation in HEK293 cells they preferentially use for their experiments.

    As suggested by the reviewer, the blots can easily be probed for p-eIF4E (S209) and pS6K1(T412) to check the effect of CGP57380 in HEK293 cells, though this has already been done in our previous manuscript (Batool et al, Molecular and Cellular Biochemistry, 2019).

    1. The quality of pS6K1(T412) blots is questionable: while on Figure 1DEF, Figure 2A, Figure 5C and Figure 7B there is a clear single band, on Figure 1G, Supplementary figure S1, Figure 5ABDEF, Figure 6CDE and Figure 7ADE the authors ignore the strong band and appear to focus on the weak one.

    The reviewer has rightly noticed the presence of one sharp band in some blots probed with Thr412 and two bands in few. The difference lies in the use of two different antibodies (Cell Signaling Technology Cat no. 9205 and 9234). One among them detects only one band while other detects two bands may be because of the potency of the antibody towards a particular species.

    1. The authors do not comment on the reproducibility nor present quantitation of the essential experiments (Figure 1EFG, Figure 3D, Supplementary figure S1, etc). Quantitation should at least include essential WBs (pS6K1(T412) and p-eIF4E(S209)) and S6K1 activity towards S6 and must explicitly state the number of independent experiments and the reported statistic.

    The quantitation for these figures can be added as suggested by the reviewer.

    1. The authors should comment on the puzzling result in Figure 1F where control shRNA significantly decreases S6K1 activity towards S6.

    We acknowledge that this is an anomaly and can be corrected.

    1. The authors should consider alternative models. Thus, for instance, Blenis lab has previously shown that S6K1 and mTORC1 cooperate in the context of eIF3 complex. Could this mechanism contribute to the increased S6K1 activity upon eIF4E overexpression?

    This possibility was over ruled as we observed a direct binding of eIF4E and S6K1.

    Furthermore, I would strongly recommend extensive editing to improve the structure and style of the manuscript.

    We agree to re-structure and re-style the manuscript as and when required.

  2. eLife

    ###Reviewer #3

    High eIF4E/4EBP1 ratio is known to predict low cell sensitivity to mTOR inhibitors, suggesting that high eIF4E could help bypass mTOR requirement for cell growth and cap-dependent mRNA translation. The manuscript by Majeed et al examines how eIF4E affects S6K1 HM phosphorylation and activity. The authors claim that phosphorylated eIF4E (and not mRaptor) is the factor required "to overcome mTORC1 dependence of S6K1" activation and suggest mTORC2 (rather than mTORC1) as a kinase phosphorylating S6K1 HM.

    To support this conclusion, the authors argue that:

    • overexpression of eIF4E WT and S209E correlates with S6K1 phosphorylation and activity and is rapamycin-insensitive (Figure 1EF, Supplementary Figure S1)

    • mTORC1 activity is required for S6K1 and eIF4E phosphorylation (Figure 2AB, Figure 3BCE)

    • S6K1 TOS, but not HM phosphorylation is required for its interaction with eIF4E (Figure 4AD)

    • MNK1 activity is not required for eIF4E phosphorylation (Figure 3CD)

    • mRaptor is not required for S6K1 binding to eIF4E (Figure 4DE)

    • mTOR is required for S6K1 activity and mTORC2 activity is responsible for phosphorylation of TOS- and CT-deficient S6K1 (Figure 5DF)

    Further, the authors identify a short peptide in S6K1, which appears to interact with PHLPP1.

    While some of the results are indeed interesting, the presented data are not sufficient to support the authors' central claim (that eIF4E and not mRaptor/mTORC1 is required for mTORC1-independent S6K1 phosphorylation and activity). Thus, the key experiment to demonstrate that (phosphorylated) eIF4E is necessary and sufficient for S6K1 phosphorylation and activity in the presence of rapamycin is missing. Figure 1F and Figure 1G come closest to that, but still fall short of convincingly supporting the central claim. Further, the fact that mTORC2 could phosphorylate the HM in TOS- and CT-deficient S6K1 has already been elegantly and definitively shown by Ali & Sabatini in their 2005 JBC publication.

    Besides the central deficiencies outlined above, the following major points should be addressed:

    1. While the authors claim that MNK1 is not the "primary" kinase phosphorylating eIF4E, they fail to show the lack of CGP57380 effect on p-eIF4E(S209) and pS6K1(T412) phosphorylation in HEK293 cells they preferentially use for their experiments.

    2. The quality of pS6K1(T412) blots is questionable: while on Figure 1DEF, Figure 2A, Figure 5C and Figure 7B there is a clear single band, on Figure 1G, Supplementary figure S1, Figure 5ABDEF, Figure 6CDE and Figure 7ADE the authors ignore the strong band and appear to focus on the weak one.

    3. The authors do not comment on the reproducibility nor present quantitation of the essential experiments (Figure 1EFG, Figure 3D, Supplementary figure S1, etc). Quantitation should at least include essential WBs (pS6K1(T412) and p-eIF4E(S209)) and S6K1 activity towards S6 and must explicitly state the number of independent experiments and the reported statistic.

    4. The authors should comment on the puzzling result in Figure 1F where control shRNA significantly decreases S6K1 activity towards S6.

    5. The authors should consider alternative models. Thus, for instance, Blenis lab has previously shown that S6K1 and mTORC1 cooperate in the context of eIF3 complex. Could this mechanism contribute to the increased S6K1 activity upon eIF4E overexpression?

    Furthermore, I would strongly recommend extensive editing to improve the structure and style of the manuscript.

  3. eLife

    ###Reviewer #2

    This manuscript builds on a previous publication from the authors identifying an mTORC1-sensitive and MNK1-mediated phosphorylation of elF4E, which they now propose is involved in the mechanism of activation of S6 kinase1 (S6K1). Specifically, the authors propose that the binding of MNK-1-phosphorylated elF4E to the TOR Signaling motif (TOS) of S6K1 relieves autoinhibition of the kinase, in turn promoting the phosphorylation by mTORC2 of the regulatory hydrophobic motif phosphorylation site. Furthermore, they propose that this phosphorylation is kept in check by binding of the phosphatase PHLPP1 to an 8 amino acid segment on S6K1, and that serum stimulation results in the release of PHLPP1 to increase phosphorylation at the hydrophobic motif and allow full activation. This is a potentially very interesting finding but unfortunately the data are poorly presented, many experiments are superficial, and alternative explanations are not considered.

    Major comments:

    1. A large portion of Figures 1-3 is a reproduction of data from the authors' 2020 paper (Batool et al., 2020) which showed that elF4E is phosphorylated by MNK1, and that MNK1 is repressed by activation of mTORC1 signaling. While some new experiments have been added (e.g. the analysis showing increased expression of S6k1 in cancer cell lines/tissue and the in silico peptide docking analysis), these are minimal additions to the recently published work from this group.

    2. One new finding in this paper is that elF4E binds the TOS motif on S6K1 and this binding promotes the hydrophobic motif phosphorylation of S6K1. The authors interpret their data to mean that binding of elF4E induces a conformational change to relieve autoinhibition. Is there any structural information to support this conformational change? What if the binding of elF4E recruits the hydrophobic motif kinase (mTORC2 proposed) in the absence of a conformational change? There are multiple other explanations that need to be considered and addressed.

    3. The authors propose that PHLPP1 is constitutively bound to S6K1 to suppress hydrophobic motif phosphorylation, and serum stimulation causes the release of PHLPP1 to fully activate S6K1. Unfortunately, this potentially important mechanism is experimentally addressed by only 3 co-IPs in Figure 7: overexpressed PHLPP1 co-IPs with a GST fusion with residues 78-85 of S6K1, PHLPP1 co-IPs with S6K1 (and less efficiently in the presence of serum), the PHLPP1 regulation of S6K1 is abolished in a construct in which residues 78-95 are deleted. The identification of a PHLPP1-binding determinant on S6K1 is significant but the current data just scratch the surface. What are the residues? Are they evolutionarily conserved? Are they conserved in other PHLPP1 substrates? Does the GST fusion with these 8 amino acids result in the activation of S6K1 by sequestering PHLPP1? A compelling mechanistic analysis is missing and should be provided especially since PHLPP1 is in the title of the paper.

    4. Deletion of residues 91- 109 inactivates S6K1, which the authors interpret as meaning the regions is critical for mTORC2 binding and HM phosphorylation. But this encompasses the Gly-rich loop and its deletion will inactivate any kinase.

  4. eLife

    ###Reviewer #1

    In this article Majeed et al propose a previously unrecognized model of S6K1 activation whereby eIF4E interacts with the TOS motif of S6K1, which facilitates phosphorylation of its hydrophobic motif by mTORC2. The authors also propose that another motif in S6K1 is responsive for serum induced and PHLPP1-mediated activation of S6K1. Furthermore, the authors propose that eIF4E may be a direct downstream substrate of mTORC1, and that mTOR is a major kinase that phosphorylates eIF4E. Although of potential interest, the data are frequently overinterpreted, the experimental design is not optimal, previous literature was not adequately considered, and many of the authors' conclusions were open to alternative explanations. My specific comments are outlined below:

    1. In many instances inappropriate controls were used. For instance, a straightforward experiment to corroborate the authors model would be to employ cells that exclusively express non-phosphorylatable eIF4E mutant (such as eIF4E KI MEFs described in Furic et al., 2010) and/or MNK KOs to establish the requirement of eIF4E phosphorylation and potential cross-talk with MNK dependent mechanisms, respectively. Although there were some attempts to do this (e.g. MNK1 KD, using pharmacological inhibitors that are by the way quite non-specific), the data are insufficient to support the authors' claims. Moreover, the interaction between eIF4E and eIF4G and potential changes in the eIF4F levels that are likely to confound authors' conclusions were not assessed.

    2. Several mechanisms involving indirect effects of mTOR on eIF4E phosphorylation that have been reported in the literature were not considered. For instance, it is plausible that mTOR affects eIF4E phosphorylation by bolstering eIF4E:eIF4G association and recruitment of MNKs.

    3. The evidence for direct phosphorylation of eIF4E by mTOR was based on non-optimally designed experiments. The description of methodology for the in vitro kinase assays was inadequate, and the experiment was carried out solely using GST-WTeIF4E as a substrate without appropriate controls. There also appears to be rapamycin dependent eIF4E phosphorylation in KD mTOR lanes.

    4. The authors use non-transformed cells as a control for eIF4E overexpression, whereby eIF4E overexpression is well-established to transform immortalized cells (Work from Sonenberg's, Bitterman's etc. labs).

    5. Functional assays are warranted to establish the effects of proposed mechanism on cell functions/fate.

    6. Many blots throughout the paper were of insufficient quality to be clearly interpreted.

    7. Many interpretations of the results were not justified by the data (e.g. in Figure 1C it is claimed that phosphorylation of eIF4E is increased in overexpressors, but this could be simply due to the increase in total protein levels).

    8. Most of the work relies on transient (except for FLAG-S6K1) overexpression strategies which are prone to artifacts and not likely to represent physiological stoichiometry of investigated proteins.

    9. It has been previously shown (e.g. Lowe & Pelletier's labs) that eIF4E confers resistance to rapamycin by mechanisms that were clearly distinct and at least in my opinion far better substantiated than those published previously by the authors and proposed here. Indeed, eIF4E overexpression results in increased eIF4F levels, which has been shown to attenuate efficacy of not just rapamycin, but also active mTOR inhibitors, and many other oncogenic-kinase inhibitors.

    10. Many published articles are misinterpreted as supporting the authors' claims. For instance, the authors write that "the inconsistent stature of mTORC1 as a 4EBP1 kinase in vivo" and the reference provided suggests that GSK3beta may phosphorylate 4E-BP1 in addition to mTOR which in certain contexts may lead to rapamycin resistance. As far as I understand, this, and other similar studies, do not challenge the status of mTORC1 as a 4E-BP1 kinase in vivo, but that GSK3beta (and other kinases such as Pim kinases, CDK1) may also phosphorylate 4E-BPs in certain contexts. Moreover, as initial studies on active-site mTOR inhibitors by Thoreen et al., and Feldman et al., as well as studies from Blenis' and Sonenberg's groups indicated, rapamycin does not efficiently inhibit 4E-BPs n the vast majority of contexts, which suggest that GSK3beta-dependent resistance to rapamycin may result from mTOR effectors other than 4E-BPs

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to Version 4 of the preprint.

    ###Summary

    While all three reviewers found this study to be conceptually of considerable interest, a number of major concerns were highlighted. Most notably, the reviewers do not feel that the central claim of the paper that phospho-eIF4E and S6K1 "interaction is sufficient to overcome rapamycin sensitivity and mTORC1 dependence of S6K1" is sufficiently supported by the evidence presented.

  6. Version published to 10.1101/2020.02.15.944892 on bioRxiv