Figure 4

mTOR can phosphorylate ERα in vitro . A) 300 ng of rh-ERα alone (full-length human ERα), or in the presence of 100 ng of mTOR (mTOR/Raptor/MLST8 complex, catalytic domain) or 100 ng of p70S6K was subjected to in vitro kinase assays in the presence of 0.2 mM ATP for 30 minutess at 30°C. Reactions were pre-incubated for 15 minutess with DMSO (vehicle control) or with selective kinase inhibitors (100 nM mTOR inhibitor AZ8055 or 10 μM p70S6K inhibitor PF-4708671). Following incubation, an aliquot of the reaction mix was subjected to Western blotting and visualized with an antibody specific for phospho-serine residues. B) 100 ng of recombinant human p70S6K was incubated with 100 ng of rh-mTOR (catalytic domain) with and without ATP as described in the Methods section. At the end of the incubation an aliquot of each reaction was subjected to Western blotting and visualized with an antibody specific for p-T389-p70S6K. C) 300 ng of rh-ERα and 100 ng of recombinant full-length p70S6K were incubated together in the presence or absence of ATP as described in the Methods section. At the end of the incubation an aliquot of each reaction was subjected to Western blotting and visualized with an antibody specific for pS167-ERα. M = molecular mass marker. DMSO, dimethyl sulfoxide; E2, estradiol; mTOR, mechanistic target of rapamycin; rh, recombinant human.