I want to find whether the test drug can activate mTORC1 but not activate mTORC2. I have checked the expression of p70s6 and 4ebp1, now, how can I check the activation of mTORC2?
you can check for phosphorylation of Akt/PKB. WB of total protein and then re-probe with a phospho ab. it is a direct target of mTORC2. Hope this helps
Since both inhibiting and activating mTORC1 can differently regulate mTORC2 activity measured by by pAkt-S473, you might want to consider whether the observed difference (if any) of pAkt-S473 is mediated by mTORC1. By the way, mTORC2-independent pAkt-S473 do exist but with very small amount, and the signal of mTORC2-independent pAkt-S473 can enhanced a lot under mTORC1 inhibition.
Based on published articles AKT-Ser473 phosphorylation is the best available way to check/predict mTORC2 activity. However, it is important to consider the comments by Dr. Tang.
@Yuefeng Tang: I found my drug can down regulate the phosphorylation of 4EBP1, and I thought that this is from mTOR inhibition, but the expression of mTOR is not changed, and p-AKT is up-regulated. There are many papers show that mTOR inhibitor can increase p-AKT by feedback. Now, I want to check the up of p-AKT is from this feedback but not from mTORC2 signaling pathway, then I can explain why p-4EBP1 is down regulated. Can you give me some advice? or what I need to do next? Thanks for your help!
@Dc Zhou: there are a few experiments that you might want to try. 1: to test whether rictor and/or mTORC2 complex (By mTOR IP) is induced by the drug, which may give you the idea whether you drug can regulate mTORC2 activity; 2: Test whether you drug can enhance pAkt-S473 in rictor or Sin1 null cells; based on my experiences using both genetic models and drug treatments, I can tell you inhibiting mTORC1 will increase pAkt-S473 in both mTORC2-dependent and -independent pattern (this might be cell line-dependent, but I believe that this should happen in almost cell line if not all); 3: does your drug inhibit pAkt-S473 at acute treatment (1 hour), but increase pAkt-S473 at prolong treatment (24 hours or longer); 4: is the enhanced pAkt-S473 blocked by mTOR Kinase inhibitor and/or PI3K inhibitor (acute treatment such as 1 hour); 5: any enhanced RTKs expression in your drug treated samples:
i would like to ask, how can we detect the expression of MTORC1 in breast cancer line, can we use the RT-PCR to see the expression instead of western blot.
You can do PCR, but western blot is better but you have to measure different components of mTORC1. If you have various cell lines, I think you might want to test the response of mTORC1 in each cell line to each stimulation since mTORC1 is in everywhere.
You can check Ser-2481 phosphorylation which is an mTORC2 specific phosphorylation and an activation marker.
mTORC2 phosphorylates Ser473 -AKT. Therefore, western blot analysis of cell extracts for detection of the p-Ser473-AKT should be done.
If you want to only focus on the mTORC2, an easy and reliable assay is to measure the expression of Akt-T450 by Western. It is true that Akt-S473 can be activated under mTORC2 activation by growth factors or RTK activation, but we can not say mTORC2 is the cause just involved. Interestingly, pAkt-T450, which is very stable, is not regulate by growth factors but require mTORC2 activity. In the Rictor or Sin1 Knockout cells or tissues, the pAkt-T450 will disappeared. BTW, it takes time for the turnover of pAkt-T450 since Torin1 can inhibit pAkt-S473 in one hour but need longer to decrease pAkt-T450.
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