Fig. 3: Insulin regulates USP5 function through mTORC1-mediated phosphorylation to enhance YTHDF1 stability.
From: USP5 stabilizes YTHDF1 to control cancer immune surveillance through mTORC1-mediated phosphorylation
a IB analysis using the indicated antibodies in PLC/PRF/5 and Hepa1-6 cells treated with increasing concentrations of insulin for 30 min. b Reduced YTHDF1 polyubiquitination upon growth factor stimulation. HEK293T cells transfected with ubiquitin constructs were lysed for anti-YTHDF1 IP and IB. c The WT and Usp5-KO cells were treated with insulin (100 ng/ml) or Torin1 (1.0 μM). After 30 min or 24 h, cells were collected and analyzed by IB. d IB analysis of YTHDF1 protein levels in PLC/PRF/5 cells expressing the indicated constructs. e Knockdown of USP5 inhibits mTOR or RPTOR-induced accumulation of YTHDF1. The WT and USP5-KD PLC/PRF/5 cells were transfected with Flag-mTOR or HA-RPTOR constructs for 36 h. f IB analysis of YTHDF1 immunoprecipitate or WCL from PLC/PRF/5 cells transfected with Flag-YTHDF1. Cells were then serum starved for 16 h and stimulated with insulin (100 ng/ml) for 30 min. g IB analysis of WCL and IP derived from HEK293T cells transfected with various HA-USP5 constructs as well as Flag-USP5. Cells were pretreated with 1.0 μM Torin1 for 24 h. h IB analysis of WCL and IP derived from HEK293T cells transfected with Flag-mTOR together with the HA-USP5 constructs for 36 h. i In vivo ubiquitination analysis of YTHDF1 in PLC/PRF/5 cells expressing the indicated HA-USP5 constructs. Cells were treated with 20 μM MG132 for 8 h. j In vivo phosphorylation assay of HA-USP5 WT and S149A mutant in HEK293T cells with or without ectopic Flag-mTOR expression. k The S149 mutant disrupted USP5 dimerization process in cells. IB analysis of WCL and IP derived from HEK293T cells transfected with constructs indicated. l Co-IP analysis of USP5/YTHDF1 interaction in HEK293T cells with expressing the indicated constructs. m A simplified model depicting the regulatory mechanism of USP5 by insulin signaling pathways. The active mTORC1 phosphorylates USP5 at S149, which promotes its dimerization and stabilizes YTHDF1, and then feedback positively regulates mTORC1 activity by increasing RPTOR mRNA translation. All data are representative of two independent experiments. Source data are provided as a Source data file.
