NFκB signaling plays a significant role in human disease, including breast and ovarian carcinoma, insulin resistance, embryonic lethality and liver degeneration, rheumatoid arthritis, aging and Multiple Myeloma (MM). Inhibitor of κB (IκB) kinase β (IKKβ) regulates canonical Nuclear Factor κB (NFκB) signaling in response to inflammation and cellular stresses. NFκB activation requires Lys63-linked (K63-linked) ubiquitination of upstream proteins such as NEMO or TAK1, forming molecular complexes with membrane-bound receptors. We demonstrate that IKKβ itself undergoes K63-linked ubiquitination. Mutations in IKKβ at Lys171, identified in Multiple Myeloma and other cancers, lead to a dramatic increase in kinase activation and K63-linked ubiquitination. These mutations also result in persistent activation of STAT3 signaling. Liquid chromatography (LC)-high mass accuracy tandem mass spectrometry (MS/MS) analysis identified Lys147, Lys418, Lys555 and Lys703 as predominant ubiquitination sites in IKKβ. Specific inhibition of the UBC13-UEV1A complex responsible for K63-linked ubiquitination establishes Lys147 as the predominant site of K63-ubiquitin conjugation and responsible for STAT3 activation. Thus, IKKβ activation leads to ubiquitination within the kinase domain and assemblage of a K63-ubiquitin conjugated signaling platform. These results are discussed with respect to the importance of upregulated NFκB signaling known to occur frequently in MM and other cancers.

Mutations at position K171 in the kinase activation loop of Inhibitor of κB kinase beta (IKKβ) occur in multiple myeloma, spleen marginal zone lymphoma and mantle cell lymphoma. Previously, we demonstrated that these result in constitutive kinase activation and stimulate Signal Transducer and Activator of Transcription 3 (STAT3). This work also identified K147 as a site of K63-linked regulatory ubiquitination required for activation of signaling pathways. We now present a more detailed analysis of ubiquitination sites together with a comprehensive examination of the signaling pathways activated by IKKβ K171E mutants. Downstream activation of STAT3 is dependent upon the activity of: UBE2N, the E2 ubiquitin ligase involved in K63-linked ubiquitination; TAK1 (MAP3K7), or TGFβ Activated Kinase, which forms a complex required for NFκB activation; JAK kinases, involved proximally in the phosphorylation of STAT transcription factors in response to inflammatory cytokines; and gp130, or IL-6 Receptor Subunit Beta which, upon binding IL-6 or other specific cytokines, undergoes homodimerization leading to activation of associated JAKs, resulting in STAT activation. We further demonstrate, using an IL-6-responsive cell line, that IKKβ K171E mutants stimulate the release of IL-6 activity into conditioned media. These results show that IKKβ K171E mutants trigger an autocrine loop in which IL-6 is secreted and binds to the IL-6 receptor complex gp130, resulting in JAK activation. Lastly, by examining the differential abundance of proteins associated with K63-only-ubiquitinated IKKβ K171E, proteomic analysis demonstrates the global activation of proliferative responses. As cancers harboring K171-mutated IKKβ are likely to also exhibit activated STAT3 and p44/42 MAPK (Erk1/2), this suggests the possibility of using MAPK (Erk1/2) and JAK inhibitors, or specific ubiquitination inhibitors. K63-linked ubiquitination occurs in other kinases at sites homologous to K147 in IKKβ, including K578 in BRAF V600E, which serves as an oncogenic driver in melanoma and other cancers.