Fig. 1
FBXL6 activates KRAS and KRASG12D by K63-linked polyubiquitination at the site K128. a Analysis of FBXL6 expression across cancers in the TCGA database shows that FBXL6 is upregulated in various types of human cancers, including liver hepatocellular carcinoma (LIHC), namely, HCC. b Alb-Cre and LSL-Fbxl6KI/+;Alb-Cre (LC) male mice were monitored for 320 d and then euthanized. The livers were imaged. c Diagram showing the number of proteins with increased ubiquitinated sites and upregulated expression in HCC tumors. Ub-C/A > 4 indicates proteins with a more than fourfold increase in ubiquitinated sites in tumors compared with normal tissues from male mice; Ub-B/A > 3 indicates proteins with a more than threefold increase in ubiquitinated sites in adjacent tissues compared with normal tissues. Ub-C/B > 1.3 indicates proteins with a more than 1.3-fold increase in ubiquitinated sites in tumors compared with adjacent tissues. TP-C/B > 1.4 and TP-C/A > 1.5 indicate that the protein levels were upregulated 1.4- or 1.5-fold in tumors compared with adjacent tissues or normal tissues, respectively. d Evolutionary conservation of the site K128 on KRAS from different species. A red star (*) indicates a conserved site in KRAS in different species. e A co-IP assay was utilized to measure the interaction between FBXL6 and KRAS. Huh7 and Hep3B cells were transfected with Flag-FBXL6 plasmids for 48 h and then lysed. The indicated antibodies and protein A/G PLUS-Agarose were added to the cell lysates. f HEK293T cells were transfected with the indicated plasmids for 72 h and then lysed with a 6 mol/L guanidine solution, followed by pull-down using Ni–NTA beads or direct Western blotting with the indicated antibodies. g Huh7 cells were transfected with the indicated plasmids for 72 h and lysed with lysis buffer. Activated KRAS was pulled down with an anti-active RAS monoclonal antibody (RBD peptide), followed by Western blotting. Band intensity was quantified by ImageJ software. h After serum deprivation for 12 h, Huh7 cells were transfected with the indicated plasmids for 72 h, followed by Western blotting. ns non‑significant; ***P < 0.001. BLCA bladder urothelial carcinoma, BRCA breast invasive carcinoma, CESC cervical squamous cell carcinoma and endocervical adenocarcinoma, CHOL cholangiocarcinoma, COAD colon adenocarcinoma, ESCA esophageal carcinoma, GBM glioblastoma multiforme, HNSC head and neck squamous cell carcinoma, KICH kidney chromophobe, KIRC kidney renal clear cell carcinoma, KIRP kidney renal papillary cell carcinoma, LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, PAAD pancreatic adenocarcinoma, PRAD prostate adenocarcinoma, PCPG pheochromocytoma and paraganglioma, READ rectum adenocarcinoma, SARC sarcoma, SKCM skin cutaneous melanoma, THCA thyroid carcinoma, THYM thymoma, STAD stomach adenocarcinoma, UCEC uterine corpus endometrial carcinoma, FBXL6 F-box and leucine-rich repeat 6, TCGA The Cancer Genome Atlas, WCE whole-cell extract, HA hemagglutinin, KRAS kirsten rat sarcoma, KRASG12D glycine to aspartic acid mutation of KRAS, Ub ubiquitin, HA-KRAS (DM) double mutation of KRAS at the sites K128 and G12, A normal liver tissue, B adjacent tumor tissue, C cancer or tumor tissue, TP total protein, K128 lysine 128, Co-IP coimmunoprecipitation, RBD RAS-binding domain