Fig. 5

HECTD3 ubiquitinates IKKα with K27- and K63-linked polyubiquitin chains at K296 and increases IKKα protein stability and kinase activity. a Flag-IKKα, HA-Ub, and HECTD3 (WT) or HECTD3 C823A were coexpressed in HEK293T cells. Ubiquitinated Flag-IKKα proteins were immunoprecipitated with Flag-M2 beads and probed with anti-HA antibody. b Co-IP analysis of the ubiquitination of endogenous IKKα in HUVECs overexpressing stable Flag-ubiquitin (Flag-Ub). The cells were transfected with siRNA to knock down HECTD3. Anti-IKKα antibody was used for immunoprecipitation. The anti-Flag antibody was used to detect ubiquitinated IKKα. c Purified recombinant HECTD3 and HECTD3 C823A proteins from E. coli were detected by Coomassie blue staining. d HECTD3 ubiquitinates IKKα in vitro in an E3 ligase activity-dependent manner. ATP, HA-Ub, E1, UbcH5b, HECTD3 or HECTD3 C823A, and Flag-IKKα were mixed for ubiquitination assays. The Flag-IKKα protein was purified from HEK293 cells transfected with the plasmid encoding Flag-IKKα using Flag-M2 beads. e HECTD3 ubiquitinates IKKα at K296. HECTD3 failed to ubiquitinate Flag-IKKα K296R, similar to WT, K311R and K322R in HEK293T cells. f HECTD3 ubiquitinates IKKα with K27- and K63-linked polyubiquitin chains. WT, K27 only, and K63 only HA-Ub supported HECTD3-mediated Flag-IKKα ubiquitination. In contrast, K33 only and K48 only HA-Ub failed to do so. g Linkage-specific antibodies were used to validate the linkage of Flag-IKKα. h CHX chase assays were used to analyze the half-lives of Flag-IKKα WT and K296R mutant in HEK293T cells. i IKKα ubiquitination at K296 is essential for LPS to induce adhesion molecule expression in HUVECs. IKKα was stably knocked out in HUVECs using the CRISPR/Cas9 system. Immunoblotting of adhesion molecule expression in these cells restored the expression of IKKα by lentivirus encoding Flag-IKKα WT or Flag-IKKα K296R and stimulated with or without LPS (300 ng/mL) for indicated time (0–4 h). j The in vitro IKKα kinase assay contains purified Flag-IKKα WT, K296R, S175/180 A, GST-H3, and ATP. Flag-IKKα proteins were purified from HEK293T cells. k HECTD3 knockdown decreased IKKα activity toward histone H3. HECTD3 knockdown and Flag-IKKα overexpression were performed in HEK293T cells. Flag-IKKα proteins were purified for in vitro kinase assays toward GST-H3. l Overexpression of HECTD3, not HECTD3 C823A, increased IKKα activity toward histone H3. Flag-IKKα and Flag-IKKα K296R proteins were purified from HEK293T cells cotransfected with plasmids encoding Flag-IKKα or Flag-IKKα K296R with HECTD3 or HECTD3 C823A. In vitro kinase assays of Flag-IKKα WT and Flag-IKKα K296R toward GST-H3 were performed. m Flag-IKKα K296R decreased the interaction with H3 compared to Flag-IKKα WT. Cell lysates of HEK293T cells expressing Flag-IKKα WT or Flag-IKKα K296R were collected and incubated with purified GST-H3 protein for 30 min on ice. The GST pull-down assay was performed using glutathione sepharose beads. Data represent three independent experiments for all of the above experiments