Novel Lys63-linked ubiquitination of IKKb induces STAT3 signaling

NFB 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 B (IB) kinase (IKK) regulates canonical Nuclear Factor B (NFB) signaling in response to inflammation and cellular stresses. NFB 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 IKK itself undergoes K63-linked ubiquitination. Mutations in IKK 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 IKK. 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, IKK 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 NFB signaling known to occur frequently in MM and other cancers.