Figure 1. Amino acid starvation stimulates a protein phosphatase for ULK1.

(a,b) Starvation induces a stronger autophagic response than rapamycin. In a, MEFs stably expressing GFP-LC3 were incubated in starvation media or treated with 1-μM rapamycin for the indicated time. Representative images from three independent experiments shown, average number of puncta per cell was quantitated and expressed as fold change relative to 0 h (fold change ±s.d., n=30. two-tail Student's t-test, *P<0.05). In b, MEFs were lysed at the indicated time points and immunoblotted for endogenous LC3 and other proteins as indicated. (c) Kinetics of mTOR substrate dephosphorylation in response to starvation or pharmacological inhibition of mTOR. MEFs were incubated in starvation media or media containing 1 μM of either Torin1 or rapamycin, and lysed at the indicated time points. Lysates were analysed for the endogenous levels and phosphorylation states of the specified proteins. (d) Torin1 shuts down mTOR activity more efficiently than starvation. MEFs were incubated in starvation media or media containing 1 μM Torin1 for the indicated amount of time. (e) Starvation increases phosphatase activity for ULK1 S637 in MEFs. Upper panel shows a schematic representation of the in vitro phosphatase assay. Middle panel shows outcome of the phosphatase assay comparing lysates from starved, fed or rapamycin-treated cells. Reactions were terminated at the indicated time and analysed by western blotting for ULK1 phosphorylation status. S-tag and total S6K are loading controls for the amount of substrate and enzyme, respectively, in each reaction. Lower panel shows quantitation of ULK1 S637 phosphorylation relative to time 0 from four independent experiments (fold change±s.d., n=4. two-tail Student's t-test, *P<0.05).