PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase
Authors: Seong A Kang
Steven A Carr
Yasemin Sancak
Robert A Lindquist
Timothy R Peterson
David M Sabatini
Eric Spooner
Carson C Thoreen
Journal: Molecular Cell (2007)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: mTORC1; PRAS40; insulin; cell growth; S6K1 phosphorylation
Affiliations: Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge, MA 02142, USA.

Abstract:  The heterotrimeric mTORC1 protein kinase nucleates a signaling network that promotes cell growth in response to insulin and becomes constitutively active in cells missing the TSC1 or TSC2 tumor suppressors. Insulin stimulates the phosphorylation of S6K1, an mTORC1 substrate, but it is not known how mTORC1 kinase activity is regulated. We identify PRAS40 as a raptor-interacting protein that binds to mTORC1 in insulin-deprived cells and whose in vitro interaction with mTORC1 is disrupted by high salt concentrations. PRAS40 inhibits cell growth, S6K1 phosphorylation, and rheb-induced activation of the mTORC1 pathway, and in vitro it prevents the great increase in mTORC1 kinase activity induced by rheb1-GTP. Insulin stimulates Akt/PKB-mediated phosphorylation of PRAS40, which prevents its inhibition of mTORC1 in cells and in vitro. We propose that the relative strengths of the rheb- and PRAS40-mediated inputs to mTORC1 set overall pathway activity and that insulin activates mTORC1 through the coordinated regulation of both. 

Retracted: No

PubPeer comments:
 Supplemental Fig. S1 Could the authors check please? More similar than expected. ![file] ![file] , 
 Fig 7B (with enhanced contrast below) shows possible splicing. Could the authors please check? *** ![file] *** Full figure ![file] , 
 There seems to be another splice in Figure 5, where the P-T389-S6K1 bands seem to have been fused from different gels, whereas the aligned S6K1 bands appear not to be. Is this intentional or a mistake? ![file] , 
 Thank you for your comments. This 13-year-old paper describes the discovery of PRAS40 as a raptor-interacting protein that inhibits mTORC1 kinase activity. In the same paper we also show that GTP-loaded Rheb directly activates mTORC1 and antagonizes PRAS40. The existence of PRAS40 as an mTORC1 inhibitor has been validated by many other labs and there is even now structural data on how it interacts with Raptor to inhibit substrate recruitment to mTORC1. Two commenters point to what appear to be a few lane splices in Figures 5E and 7B. As I have explained for similar comments regarding some of our other old papers, we would have intentionally made the splices because other samples were run in between the ones we wished to show in the figure. Different gels would have been run for each of the blots shown, some of which may have needed to be spliced and others not, depending on when in the construction of the figure the gels were run. In addition, not all blots will have splice junctions because often we re-ran the gels for certain blots because there were issues with the processing of the gel or the immunoblot, which provided an opportunity to analyze only the samples relevant to the final figure. As was common practice in 2007, we did not indicate the splice junction with a line. I think it is important to point out that the presence of a lane splice has no impact on the validity of the data shown or our conclusions, which have been validated by others. Best wishes, David M. Sabatini , 
 In response to Calcinus Nitidus on the potential duplication of the S6K1 loading control in Supplemental Figure S1, at first I agreed with you that the the two pairs of bands look quite similar but then I put them through an image analysis service and it indicated that they are in fact different. With closer inspection I too started to see the differences by eye. For example, the left band in the leucine stimulation experiment is flatter than the corresponding band in the insulin stimulation experiment. The small white clearing below the left most band is also in a different position in the two images. Next to the small white clearing in the insulin stimulation experiment there is an even smaller clearing that is not present in the leucine stimulation experiment. I will also point out that these control experiments simply show that S6K1 phosphorylation is regulated by leucine as well as insulin, findings first made by others and literally duplicated by 100s of paper, including the one in question. We used the following site to compare the images: https: online-image-comparison.com best wishes, David M. Sabatini , 
 #5 Prof. Sabatini, It is true that there are some minor differences between the two panels, but the same is also true for the lettering at the top of the figure (e.g., HEK-293E), which surely were originally the same, indicating that the figure as a whole likely acquired some minor image degradation during subsequent file processing (e.g., compression, downsampling, etc.). For such reasons, pixel-for-pixel identity is not the appropriate standard to use when judging whether two similar images have a shared origin. If interested, an extensive discussion of this issue can be found in the following affidavit from John Krueger, former ORI official; see points 15, 17, 18, 20 on pgs 8-10 ( https: www.aclu.org sites default files assets krueger_affidavit.pdf ). There is also a demonstration of how identical images can acquire differences during lossy file saving at the following PubPeer thread: https: pubpeer.com publications CB23306078D45C44C2E2F6E556506D#47 None of this contests your point about the subsequent reproducibility of the findings, but merely suggests caution in interpreting minor pixel differences as evidence of distinct origins. For reference, below are the panels in question, along with text from the top, taken from the Supp PDF (captured after zooming to 1200%). (Click to enlarge.) To me, it seems the possibility of a shared origin is more likely than not. Obviously, original image files could clarify the matter more convincingly. ![file] , 
 Thanks for clarifying and point taken. When I am back in the office we will retrieve the original scans and I will provide an update. But I agree that they certainly look very similar. best, david , 
 #6 As requested, I went through my old files and oddly enough the Adobe Illustrator file marked on my computer as Figure S1 has the correct total S6K1 blots for the leucine and insulin stimulation experiments. The files for the blots are linked in the Illustrator file and so I can still click on the blots and see their file names which are different and correct (288A_293E_ins_tS6K vs 288A_293E_leu_tS6K). So, the error must have happened when we generated the Supplemental file in Word. I suspect that when the Illustrator file was placed into the Word document, the links for the blot files were lost and we had to relink them and the error happened then. Below is submitted file for Figure S1. Thanks for catching this error in the supplemental file. ![file] , 
 I am glad that the authors found that there was indeed a duplication in the published version of Figure S1, after first denying there was. Are the authors planning to issue an Erratum to correct this duplication? It s been two years since this discussion, but I cannot find any update on the official paper s landing page. , 
 Fig. 2E - Number of bands do not match. Could authors check please? ![file] , 
 Fig. 7A: Possible differential splice, could authors check please? ![file] ,