Sin1 phosphorylation impairs mTORC2 complex integrity and inhibits downstream Akt signalling to suppress tumorigenesis
Authors: Byeong Mo Kim
Min Yuan
Wenjian Gan
Dou Liu
Bo Zhai
Bing Su
Hiroyuki Inuzuka
Adam S Lazorchak
Lixin Wan
Brendan D Manning
Pengda Liu
John Blenis
Tae Ho Lee
Daming Gao
Yonghao Yu
Shavali Shaik
John M Asara
Omotooke Arojo
Suchithra Menon
Steven P Gygi
Wenyi Wei
Journal: Nature Cell Biology (2013)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: mTOR; mTORC1; mTORC2; Sin1; phosphorylation
Affiliations: Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.

Abstract:  The mechanistic target of rapamycin (mTOR) functions as a critical regulator of cellular growth and metabolism by forming multi-component, yet functionally distinct complexes mTORC1 and mTORC2. Although mTORC2 has been implicated in mTORC1 activation, little is known about how mTORC2 is regulated. Here we report that phosphorylation of Sin1 at Thr 86 and Thr 398 suppresses mTORC2 kinase activity by dissociating Sin1 from mTORC2. Importantly, Sin1 phosphorylation, triggered by S6K or Akt, in a cellular context-dependent manner, inhibits not only insulin- or IGF-1-mediated, but also PDGF- or EGF-induced Akt phosphorylation by mTORC2, demonstrating a negative regulation of mTORC2 independent of IRS-1 and Grb10. Finally, a cancer-patient-derived Sin1-R81T mutation impairs Sin1 phosphorylation, leading to hyper-activation of mTORC2 by bypassing this negative regulation. Together, our results reveal a Sin1-phosphorylation-dependent mTORC2 regulation, providing a potential molecular mechanism by which mutations in the mTORC1-S6K-Sin1 signalling axis might cause aberrant hyper-activation of the mTORC2-Akt pathway, which facilitates tumorigenesis. 

Retracted: No

PubPeer comments:
 Figure 2e. Much more similar than expected.  , 
 2019 author correction. https: pubmed.ncbi.nlm.nih.gov 30783264 The 2019 author correction does not mention figure 2. , 
 #1 After receiving the comment, we immediately approached Dr. Pengda Liu, the first author of this 2013 Nature Cell Biology paper. We found out that indeed these blank blots for mTOR and Rictor presented in the original Figure 2e look similar. We referred to the original blots that have been included in Fig. S8 in the published paper, and located the original scanned images. Given that there is no signal in these blank blots, we failed to find this mistake at the time of submission. To correct it, we presented below the original uncropped immunoblot images that were used to generate the original Figure 2e (which has also been published in Figure S8 in the 2013 NCB paper in the supplemental material section). We also used the newly cropped image derived from the original blot to generate a corrected Figure 2e shown below. tAs indicated in the original uncropped blots shown below, we performed the gel filtration experiments to examine complex formation ability for indicated HA-Sin1-WT and HA-Sin1-EE mutant. Collected cell lysates were run on SDS-PAGE gels and transferred onto the same PVDF membrane for immunoblot analysis, therefore the expression levels of indicated proteins at different fractions can be compared to one another of each immunoblot with the same exposure time on the same film. Given that no mTOR nor Rictor signal was observed in these western blots, we mistakenly used the same blank blot twice for both Rictor and mTOR. Due to the fact that these are blank blots, we failed to identify this error during manuscript preparation. Thanks for catching this error and we deeply apologize that this careless mistake was not identified before publication, even with the inclusion of original scans for these blots as shown in published supplemental Figure S8. However, this mistake does not affect the conclusions of this report.  , 
 I fully agree with the response from Dr. Wenyi Wei and sincerely apologize for this mistake due to similarities between blank blots. , 
 I fully agree with the response from Dr. Wenyi Wei and sincerely apologize for this mistake due to similarities between blank blots. , 
 #3, #5 Thank you for your replies. Could the authors please provide a high resolution version of the original uncropped scans? You may post those here or use a link to an external site (such as Imgur.com or Google Drive) for larger files. , 
 The 2013 correction mentions figures 3b and 9. Figure 3c. Much more similar than expected.  , 
 Figure 7d. Much more similar than expected.  , 
 #3  Given that there is no signal in these blank blots, we failed to find this mistake at the time of submission. Please look inside the yellow rectangles. There does seem to be quite high resolution signal.  , 
 Please find the original scans for Fig. 2e with a higher resolution as below.  , 
 This is Pengda Liu, the first author of this 2013 Nature Cell Biology paper. We found out that indeed the Rictor blot in Flag-IP was inadvertently also included in the WCLs. Attached is the corrected Fig. 3c, in which the correct Rictor blots in WCLs are included. We also present below the original uncropped immunoblot images for Rictor blots in WCLs. Thanks for catching this error and we deeply apologize that this careless mistake was not identified before publication. In addition, we also immediately repeated this experiment with a similar observation. We sincerely apologize for this mistake.  , 
 #10 Could the authors please compare the u201cblanku201d bands in the the published images with the u201coriginalu201d data? There appears to be scratches and other markings on the duplicates in the paper that are not present in the original. ***  ,