Fig. 2: LYMTACs induce ubiquitylation and lysosomal degradation of KRASG12D.
A Schematic of the chemical genetic system where the target protein is HiBiT-FKBP12F36V-KRASG12D, and the effector is MTH1-RNF152. B Structure of LYMTAC-2 (FKBP12F36V ligand-PEG2-MTH1 ligand). C HiBiT cellular degradation assay in knock-in HCT116 (HiBiT- FKBP12F36V-KRASG12D: (HF-KRAS)) cells in the absence or presence of stably expressing MTH1-RNF152. Cells were treated with increasing concentrations of LYMTAC-2 for 24 h, and HiBiT levels were measured using Nano-Glo® HiBiT lytic reagent. Data are representative of three independent experiments, reported as the mean ± S.E. D HCT116 (HF-KRAS) cells stably expressing FLAG-MTH1-RNF152 were pre-treated with BafA1, MG-132, or TAK-243 for 30 min followed by co-treatment with DMSO or 500 nM LYMTAC-2 for 6 h and subjected to immunoblotting with KRAS and tubulin antibodies. Quantified data are representative of two independent experiments. E KRASG12D ubiquitylation assay. HA-ubiquitin was transfected into HCT116, pre-treated with 200 nM BafA1 for 30 min, then co-treated with DMSO or 1 µM LYMTAC-2 for 4 h. Next, cells were lysed and subjected to immunoprecipitation with anti-HiBiT antibody. Whole cell lysate was used as the input. The respective blots are probed with HA, MTH1, HiBiT, and vinculin antibodies. Data are representative of three independent experiments.
