Figure 2. LARP1 is a direct substrate of mTOR, Akt, and S6K1.
(A) Schematic position of LARP1 phosphorylation sites identified by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). (B) Location and sequence conservation of LARP1 phosphorylation sites. (C–E) S6K1 (C), Akt (D), and mTOR (E) directly phosphorylate LARP1 in vitro. In vitro kinase assay (IVK) were performed with the indicated wild type kinase (WT) and inactive kinase (KD) purified from HEK293T cells using the indicated GST-LARP1 fragments. (F–G) Active S6K1 (F) or Akt (G) enhances phosphorylation of wild-type LARP1 but not the S770A/S979A LARP1 mutant in HEK293T cells. Phosphorylation of LARP1 was detected by phospho-specific-Akt substrate antibody. (H) Levels of LARP1 phosphorylation sites of AGC kinases are enhanced by amino acids or amino acids/growth factors. (I) Amino acids/growth factors-inducible S770/S979 phosphorylation of LARP1 is partially inhibited by rapamycin but largely inhibited by Torin1 or MK-2206. HEK293T cells were serum starved over night and incubated with HBSS with or without the indicated inhibitors for 1 hr before stimulation with DMEM containing 10% FBS for 10 min. (J) Levels of S770/S979 phosphorylation of LARP1 are decreased by rapamycin or S6K1 inhibitor (PF 470861) and further decreased by Torin1 or MK-2206 under steady state growth conditions.
Figure 2—figure supplement 1. LARP1 is a direct substrate of mTOR, Akt, and S6K1.
