Fig. 1: mTORC1 phosphorylates VAMP8 at T48.

A The extent of VAMP8 phosphorylation decreased upon inhibition of mTORC1. Protein lysates were prepared from HEK293T cells cultured in rich medium (untreated), EBSS medium, or that treated with Torin1 or rapamycin. Phosphorylated VAMP8 was blotted using antibodies against anti-phospho-Ser/Thr. Actin, LC3, p62, S6K, and p-S6K were detected by the indicated endogenous antibodies. B Nutrient-stress-induced VAMP8 dephosphorylation in a time-dependent manner. HEK293T cells were stimulated with EBSS for 15, 30, or 60 min or cultured with complete medium for 2 h after 1 h starvation. The VAMP8 phosphorylation level was detected using anti-phospho-Ser/Thr antibodies. 4EBP1, p4EBP1, S6K, pS6K were detected by the indicated endogenous antibodies. C Fasting decreased VAMP8 phosphorylation in mice liver. Mice were fed with normal diet (Fed), fasted, or refed after fasting. Western blot was employed to analyze the indicated protein levels. D Raptor knockdown inhibits VAMP8 phosphorylation. Raptor expression was inhibited by transfection of HEK293T cells with anti-Raptor siRNA. VAMP8 phosphorylation level was detected by western blot. E mTORC1 associates with VAMP8 in a nutrient-depended manner. HEK293T cells were transfected Flag-VAMP8, and the interaction of Flag-VAMP8 and mTORC1/Raptor was analyzed by immunoprecipitation under full medium or EBSS/Torin treatment. The mTOR/Raptor level was analyzed by anti-mTOR/Raptor antibody. F mTORC1 phosphorylates VAMP8 T48 in vitro. Flag-VAMP8^WT/VAMP8^T48A/VAMP8^2A/VAMP8^S55A recombinant protein was purified from E. coli and incubated with purified mTORC1 or an mTOR^D2357E (Kinase Dead) mutant. The phosphorylation level was detected by γ-³²P-ATP autoradiography. G The specificity of the antibody for the T48-phosphorylated form of VAMP8. Flag-VAMP8^WT/VAMP8^T48A proteins were pulled down using Flag beads and incubated with purified mTORC1 or mTORD^2357E. The VAMP8 phosphorylation level was detected with anti-phospho-VAMP8(T48) antibodies. H Vector control or Flag-VAMP8^WT/VAMP8^T48A was co-expressed with either Myc-tagged mTORC1 or mTOR^D2357E in HEK293T cells. Twenty-four hours after transfection, Flag-tagged proteins were purified by Flag beads and the VAMP8 phosphorylation level was detected by anti-phospho-VAMP8(T48) antibodies.