IKKβ constructs.

(A) WT Constructs. Multiple sites of ubiquitination in IKKβ were identified by LC/MS-MS which were removed by site-directed mutagenesis. Initially, the IKKβ 4KR mutant was constructed with the mutations K301R, K418R, K555R, and K703R. Further analysis revealed additional ubiquitination sites, of which five more were mutated to create IKKβ 9KR with the additional mutations K310R, K428R, K509R, K614R, and K641R. (B) K171E Constructs. The kinase activating mutation K171E was introduced into the constructs shown in (A) to create IKKβ K171E, IKKβ K171E 4KR, and IKKβ K171E 9KR. (C) K147R/K171E Constructs. The mutation K147R, identified as a major site of K63-linked ubiquitination and required for IKKβ kinase activity [5], was introduced into the constructs shown in (B) to create IKKβ K147R/K171E, IKKβ K147R/K171E 4KR, and IKKβ K147R/K171E 9KR. The ubiqutin-like domain (ULD), the scaffold/dimerization domain (SDD) which contains the leucine zipper (LZ) and helix-looop-helix (HLH) regions, and NEMO binding domain (NBD) are indicated.