Fig. 7
Trabid is an AKT substrate and this phosphorylation activates the DUB activity of Trabid. a Putative Akt phosphorylation sites predicted by Scansite within Trabid orthologs. b1 Electrospray ionization mass spectrometry (ESI-MS) analysis reveals the levels of S78 and Thr117 phosphorylation in Trabid from HEK293T cells. b2 Endogenous Trabid in Pten knockout mouse embryonic fibroblasts (MEFs) cells treated with or without AKT inhibitor were immunoprecipitated and probed with phospho-Ser/Thr antibody. b3 The phosphorylation of Trabid was found using a phospho-Ser/Thr antibody. b4 HEK293T cells were transfected with wild-type or mutant Trabid constructs, then phosphorylation of Trabid was detected using the phospho-Ser/Thr antibody. b5 The level of phosphorylated Trabid from HEK293T cells treated with or without λ-phosphatase. c1 Recombinant Trabid protein (1 μg) was mixed with or without active AKT (1 μg) in vitro, and Ub-AMC assay was performed. Relative fluorescence units (RFU). c2, c3 AKT activates Trabid in cells. Trabid was immunoprecipitated from HEK293T cells coexpressed with activated AKT (c2) or treated with 10 μM MK2206 (c3), and followed by Ub-AMC hydrolysis assay. c4 S78A/T117A mutation blocks Trabid activation by AKT. d Trabid−/− MEF cells were virally transfected with indicated plasmids, and the K63-Ubiquitination of Twist1 was analyzed. e Trabid−/− MEF cells were virally transfected with indicated plasmids with or without Myr-Akt, and the K63-Ubiquitination of Twist1 was analyzed. f Trabid−/− HCC cells were virally transfected with indicated plasmids with or without 1 µM MK2206, and the K63-Ubiquitination of Twist1 was analyzed
