Figure 1. RelA ubiquitination is promiscuous and involves non-degradative polyubiquitin chains.
(A) Ubiquitination of truncation mutants of RelA. Gal4-tagged RelA was expressed together with His-tagged ubiquitin in HEK293 cells. After lysis of the cells, ubiquitinated proteins were precipitated with Ni-NTA beads and this material was immunoblotted for RelA using either amino-terminal (F-6) or carboxyl-terminal (C-20) specific antibodies against RelA. Recovery of polyubiquitinated proteins was confirmed by immunoblotting for ubiquitin (P4D1). The non-ubiquitinated RelA proteins are indicated by arrows and non-specific bands are noted by asterisks. PD: pull-down; FL: full-length RelA (amino acids 1–551); RHD: Rel homology domain (amino acids 1–285); TAD: transactivation domains (amino acids 286–551). (B) RelA ubiquitination involves K63 and non-K63 linked polyubiquitin chains. RelA ubiquitination was examined as in (A) after expression of a ubiquitin mutant where all lysines except K63 had been mutated to arginine (K63 only), or after expression of a ubiquitin mutant where only K63 had been mutated to arginine, leaving all other lysines intact (K63R). RelA was detected using an HA antibody. (C) Ubiquitination of RelA by non-K48 and non-K63 linked polyubiquitin chains. U2OS cells were cultured with Doxycycline for 4 days to express Tet-inducible shRNAs against all 4 human ubiquitin genes and Tet-inducible HA-tagged ubiquitin (wild-type, WT, or the lysine mutants K63R or K48R). After 3 h treatment with MG-132 (20μM), denaturing cell lysates were prepared and RelA immunoprecipitation was performed. The presence of ubiquitinated RelA was determined by immunoblotting for ubiquitin or HA. Similarly, ubiquitinated RelA forms were detected after short and long exposures of the RelA immunoblots. (D) RelA is K63-polyubiquitinated after TNF stimulation. HEK293 cells transfected to express His-tagged RelA and HA-tagged ubiquitin were stimulated with TNF (2000 u/mL for 60 min) and then lyzed. RelA was precipitated (Ni-NTA beads) and the material was probed with HA antibody or a K63-specific polyubiquitin antibody. (E) Only complete deficiency of lysines prevents RelA ubiquitination. Lysine residues in RelA were progressively mutated to arginine (K/R) in the amino terminus (N, amino acids 1–218), the carboxyl terminus (C, amino acids 301–551) or the entire RelA polypeptide (All, amino acids 1–551). RelA ubiquitination was assessed as in (B).