Fig. 2.

Phosphorylations of Ulk1 at serine 638 and serine 758 were mediated differently by mTOR and AMPK. (A) Rapamycin treatment induced dephosphorylation of Ulk1 at both serine 638 and serine 758. U2OS cells were cultured in rich medium and rapamycin was added (100 nM). Cells were then collected at given time points after rapamycin treatment. (B) Knockdown of mTOR induced dephosphorylation of Ukl1 at both serine 638 and serine 758. U2OS cells were transfected with either control siRNA (Luc) or mTOR siRNA oligos. 72 h after transfection, cells were collected and extracts analyzed by Western blotting. (C) Knockdown of AMPK induced dephosphorylation of Ulk1 at serine 638, but not serine 758. U2OS stable cell lines that inducibly knock down AMPKα (α1 and α2) or AMPKβ (β1 and β2) upon addition of doxycycline (Dox) were generated. 72 h after addition of Dox, phosphorylations of Ulk1 at Ser638 and Ser758 were visualized by Western blotting. (D) AMPK activity correlates with phosphorylation of Ulk1 at serine 638 but not serine 758 in response to various nutrient conditions. U2OS cells were treated for 2 h as indicated and cell extracts were analyzed by Western blotting.