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Oncogenic mutations in IKK? function through global changes induced by K63-linked ubiquitination and result in autocrine stimulation.

ABSTRACT: Mutations at position K171 in the kinase activation loop of Inhibitor of ?B kinase beta (IKK?) 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 IKK? 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 TGF? Activated Kinase, which forms a complex required for NF?B 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 IKK? K171E mutants stimulate the release of IL-6 activity into conditioned media. These results show that IKK? 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 IKK? K171E, proteomic analysis demonstrates the global activation of proliferative responses. As cancers harboring K171-mutated IKK? 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 IKK?, including K578 in BRAF V600E, which serves as an oncogenic driver in melanoma and other cancers.

SUBMITTER: Meyer AN

PROVIDER: S-EPMC6193727 | biostudies-other | 2018

REPOSITORIES: biostudies-other

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Oncogenic mutations in IKKβ function through global changes induced by K63-linked ubiquitination and result in autocrine stimulation.

Meyer April N AN Gallo Leandro H LH Ko Juyeon J Cardenas Guillermo G Nelson Katelyn N KN Siari Asma A Campos Alexandre R AR Whisenant Thomas C TC Donoghue Daniel J DJ

PloS one 20181018 10

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 ...[more]

PMID: 30335863

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Project description:Viruses have adopted diverse strategies to suppress antiviral responses. Hepatitis B virus (HBV), a virus that is prevalent worldwide, manipulates the host's innate immune system to evade scavenging. It is reported that the hepatitis B e antigen (HBeAg) can interfere with NF-?B activity, which then leads to high viral loads, while HBV with the G1896A mutation remains infectious without the production of HBeAg but can induce more severe proinflammatory response and liver damage. The aim of current work was to study the molecular mechanism by which HBeAg suppresses interleukin-1? (IL-1?)-stimulated NF-?B activity, which leads to the suppression of the innate immune responses to HBV infection. Our study revealed that HBeAg could interact with NEMO, a regulatory subunit associated with I?B kinase, which regulates the activation of NF-?B. HBeAg suppressed the IL-1?-induced tumor necrosis factor (TNF)-associated factor 6 (TRAF6)-dependent K63-linked ubiquitination of NEMO, thereby downregulating NF-?B activity and promoting virus replication. We further demonstrated the inhibitory effect of HBeAg on the NF-?B signaling pathway using primary human hepatocytes, HBV-infected HepG2-NTCP cells, and clinical liver samples. Our study reveals a molecular mechanism whereby HBeAg suppresses IL-1?-induced NF-?B activation by decreasing the TRAF6-dependent K63-linked ubiquitination of NEMO, which may thereby enhance HBV replication and promote a persistent infection.IMPORTANCE The role of HBeAg in inflammatory responses during the infection of hepatitis B virus (HBV) is not fully understood, and several previous reports with regard to the NF-?B pathway are controversial. In this study, we showed that HBeAg could suppress both Toll-like receptor 2 (TLR2)- and IL-1?-induced activation of NF-?B in cells and clinical samples, and we further revealed novel molecular mechanisms. We found that HBeAg can associate with NEMO, the regulatory subunit for I?B kinase (IKK) that controls the NF-?B signaling pathway, and thereby inhibits TRAF6-mediated K63-linked ubiquitination of NEMO, resulting in downregulation of NF-?B activity and promotion of virus replication. In contrast, the HBeAg-negative HBV mutant can induce higher levels of NF-?B activity. These results are important for understanding the HBV-induced pathogenesis of chronic hepatitis and indicate that different clinical measures should be considered to treat HBeAg-positive and HBeAg-negative infections. Our findings represent a conceptual advance in HBV-related suppression of NF-?B signaling.

Dataset Information

Oncogenic mutations in IKK? function through global changes induced by K63-linked ubiquitination and result in autocrine stimulation.

Publications

Oncogenic mutations in IKKβ function through global changes induced by K63-linked ubiquitination and result in autocrine stimulation.

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