Fig. 3: Exo70 is phosphorylated by ULK1 on Ser47, Ser59, and Ser89.

a The level of threonine/serine phosphorylation of endogenous Exo70 under starvation conditions or rapamycin treatment. MDA-MB-231 cells were placed in starvation medium (EBSS, HBSS, or DMEM medium sugarless) or treated with rapamycin (50 nM) for 2 h, and then subjected to immunoprecipitation using anti-Exo70 antibody. Whole-cell lysates (WCLs) and immunoprecipitated (IP) proteins were analyzed by western blotting. Threonine/serine phosphorylation on Exo70 was detected by anti-p-Ser/Thr antibody. b 293T cells were transfected with empty vector or HA-ULK1 plasmid, and immunoprecipitation assay was carried out with anti-Exo70 antibody. Overexpression of ULK1 (HA-ULK1) increased the threonine/serine phosphorylation of endogenous Exo70 protein. c In vitro phosphorylation of Exo70 by ULK1. Bacterially purified Exo70 was incubated with purified HA-ULK1 and HA-ULK1(M92A) in the in vitro phosphorylation assay as described in Materials and methods. d Potential ULK1-phosphorylated sites within the N terminus of Exo70 based on the consensus ULK1 phospho-substrate sequence. e Effects of Exo70 mutations on its phosphorylation induced by ULK1. HA-ULK1 was co-transfected with the indicated FLAG-Exo70 plasmids in 293T cells, and then subjected to immunoprecipitation with anti-Flag antibody. Phosphorylation of Exo70 at serine/threonine was examined with p-Ser/Thr antibody. f Ser89 was identified to be another potential ULK1-phosphorylated site using immunoprecipitation in combination with mass spectrometry (IP-MS). g In vitro phosphorylation of wild-type and mutant Exo70 by purified ULK1. Exo70 at serine/threonine was examined with p-Ser/Thr antibody.