title: Novel Lys63-linked ubiquitination of IKKβ induces STAT3 signaling.
abstract: 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.
authors.affiliation: a Department of Chemistry and Biochemistry ; University of California San Diego ; La Jolla , CA USA.
authors.forename: Leandro H
authors.initials: LH
authors.lastname: Gallo
authors.forename: April N
authors.initials: AN
authors.lastname: Meyer
authors.forename: Khatereh
authors.initials: K
authors.lastname: Motamedchaboki
authors.forename: Katelyn N
authors.initials: KN
authors.lastname: Nelson
authors.forename: Martin
authors.initials: M
authors.lastname: Haas
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Donoghue
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D057809:HEK293 Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D010455:Peptides / Q000032:analysis; D010766:Phosphorylation; D011485:Protein Binding; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D014157:Transcription Factors / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D016328:NF-kappa B; D010455:Peptides; D050796:STAT3 Transcription Factor; D014157:Transcription Factors; C471607:UBE2N protein, human; C471624:UBE2V1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D051550:I-kappa B Kinase; D008239:Lysine
keywords: IKKβ; K-63 ubiquitination; NFκB; STAT3; cell proliferation; inflammatory signaling; mass spectrometry; multiple myeloma; oncogenesis; polyubiquitination