Hallo. I am not an expert in the topic, but I just bumped into AlphaScreen® SureFire®

mTOR (p‐Ser2448) Assay Kits from TGR Biosciences/Perkin Elmer

http://www.perkinelmer.com/Catalog/Family/ID/AlphaScreen%20SureFire%20PhosphomTOR%20Ser2448

I sincerely hope that you will find something suitable

To my knowledge, there are no reliable tools for such purpose available. I would certainly not rely on mTOR S2448 phosphorylation status. This phosphosite is not well understood or characterized, and I would not rely on any readout that is not functionally understood.

The 4EBP, S6 or S6K phosphorylations are well characterized readouts for mTORC1, while Akt S473 is a readout for mTORC2. There are certainly fluorescence based assays available that circle around those targets. If you'd ask me though, I would rather focus on a more functional readout for a small molecule inhibitor screen when dealing with mTOR. For example, we utilized a cell line that is very sensitive to mTORC2 inhibition for a full genome shRNA screen (Colombi, M., Molle, K. D., Benjamin, D., Rattenbacher-Kiser, K., Schaefer, C., Betz, C., Thiemeyer, A., et al. (2011). Genome-wide shRNA screen reveals increased mitochondrial dependence upon mTORC2 addiction. Oncogene, 30(13), 1551–1565. doi:10.1038/onc.2010.539)

I thank George Komis & Charles Betz for your effort and suggestions. I got some inputs from the above papers. Hope I will come to some conclusion once I finish reading it.

I agree, I would not consider mTOR Ser2448 autophosphorylation to be a reliable indicator of mTOR inhibitor activity as this site has been shown in some papers to be mTOR-dependent and in others non-mTOR-dependent, which could ultimately confuse your results.

In my personal experience, both the immediate downstream kinase and its kinase substrate (p70 S6 kinase and S6, respectively) will give consistent and reliable readouts of mTORC1 activity; in this regard, pS6K (Thr389) and pS6 (Ser235/236) are the two best sites to look at although certain phospho-sites on 4E-BP1 can also be utilized. If you are screening to inhibit mTOR kinase activity in general, mTORC2 activity can be actively monitored by looking at the pAkt (Ser473) site.

Positive inhibitor controls for mTORC1 and mTORC1 + mTORC2 activity would be rapamycin (2nM overnight or 20nM for 5 - 7h in most cell lines) and WYE-132, respectively. Of note is the observation of a rapamycin-mediated feedback mechanism (via IRS-1) that results in the activation of pAkt (Ser473) in some cell types upon prolonged rapamycin exposure, which should be taken into consideration in your assays;

http://sabatinilab.wi.mit.edu/Sabatini%20papers/mTORC2_rapa-MCell-2006.pdf

With respect to fluorescence-mediated monitoring of mTOR inhibition you might want to look into the LANCE Ultra ULight p70 S6K and 4E-BP1 kits, which are TR-FRET-based kinase assays;

http://www.perkinelmer.com/Catalog/Category/ID/LANCE%20Ultra%20Acceptor%20dye%20conjugates%20Kinase%20assay%20ULightlabeled%20substrate%20reagents

Alternative TR-FRET kinase assay kits include the LanthaScreen kits by Invitrogen;

http://tools.invitrogen.com/content/sfs/posters/0407-SBS-mTOR-Protein-and-Assay.pdf

Other potential screening options are the K-LISA kits from EMD Biosciences;

http://www.emdmillipore.com/life-science-research/k-lisa-mtor-activity-kit/EMD_BIO-CBA055/p_uuid?ProductID=056b.s1Oh64AAAEjskBHkR1L

Cell Signaling also have the fluorescent-based PathScan assays, which would allow you to look at both mTORC1 and mTORC2 activity, via p70 S6K (Thr389) and pAkt (Ser473) respectively;

http://www.cellsignal.com/products/7744.html

However, given the expense and broader pathway coverage that these PathScan assays offer I would probably use this as a final step to look at off-target effects of mTOR inhibitors based on output generated from more focused, cost-effective assays as listed above.

I hope this information helps and good luck with your research!

Cheers,

Jonathan