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Activating mutations of Lys171 in the kinase domain of IKKbeta unleash a novel mechanism of oncogenic signaling


Inhibitor of kappaB kinase beta (IKKbeta) is the master regulatory kinase that modulates canonical nuclear factor kappaB (NFkappaB) inflammatory activation. Under inflammatory conditions, IKKbeta is phosphorylated at Ser177/Ser181 sites in the kinase domain, which activates the kinase function of the protein. IKKbeta, in turn, phosphorylates its downstream substrate IkappaBalpha, which is subsequently degraded. This event releases NFkappaB, which is normally sequestered in the cytoplasm, to translocate into the nucleus to activate transcription of inflammation-related genes.

In the first chapter of this dissertation, mutations of Lys171 located in the kinase domain of IKKbeta are shown to lead to increase the phosphorylation of Ser177/Ser181 sites and result in the upregulation of the kinase activity of IKKbeta. These mutations had been initially identified in tumor samples of Multiple Myeloma, Spleen Marginal Zone Lymphoma and Mantle Cell Lymphoma. Via site-directed mutagenesis, we constructed the K171E mutation in IKKbeta. This study further demonstrated that IKKbeta is conjugated to Lys63-linked ubiquitin chains, in which the activating K171E mutation upregulated this modification at Lys147. The main discovery of this project is that the K171E mutation in IKKbeta induces the activation of STAT3 in the absence of exogenous Interleukin-6 in HEK293T cells. Via LC-MS/MS, we identified various lysine ubiquitination sites in full length activated IKKbeta, in which some of these negatively regulate the phosphorylation of Ser177/Ser181. Ubiquitination of Lys147 in activated IKKbeta, located in proximity to the activation loop in the kinase domain, is critical to induce STAT3 activation.

The second chapter of this dissertation demonstrates that IKKbeta K171E relies on JAK, gp130, and TAK1 to signal to STAT3. In addition, we utilized a method termed Bio-ID proximity dependent biotinylation to identify proteins interacting with activated IKKBeta, in which UBC13 and LRPPRC were identified as interacting partners. Via siRNA, we showed that UBC13 and LRPPRC possibly negatively regulated STAT3 activation induced by IKKbeta K171E. Interestingly, IKKbeta K171E depends upon UBE2V2 cofactor of UBC13, previously demonstrated to be involved in modulation of DNA damage repair activation, to activate STAT3. Lastly, the oncogenic potential of K171E mutation is demonstrated by the transformation of murine myeloid 32D cells into Interleukin-3-independent phenotype. In conjunction, these results illustrate a novel mechanism by which the loss of Lys171 in IKKbeta induces the activation of STAT3 in the absence of cytokines and growth factors.

The third chapter of this dissertation entails a review article describing that the misregulated expression of members of the ubiquitination cascade contributes to cancer progression and metastasis. Ubiquitination largely serves as a degradation mechanism of proteins. Ubiquitin signaling is further involved in additional cellular processes such as the activation of NFκB inflammatory response and DNA damage repair. In this review, we highlight the E2 ubiquitin conjugating enzymes, E3 ubiquitin ligases and Deubiquitinases that support the proliferation, migration, stemness and metastasis of a plethora of cancers, including their contribution to poor clinical prognosis and modulation of pluripotent cancer stem cells attributed to metastasis. We further describe mutations in E3 ubiquitin ligases that support the proliferation and adaptation to hypoxia of some cancers. Thus, this review illustrates how tumors exploit the members of the vast ubiquitin signaling pathways to support oncogenic signaling for the purposes of survival and metastasis.

The fourth chapter of this dissertation entails a review article related to mutations in Fibroblast Growth Factor Receptors (FGFRs) in cancer and developmental disorders. This comprehensive review describes approximately 200 different mutations in FGFRs in diverse cancers, in which some of these mutations have been found to contribute to developmental syndromes. In addition, this review shows the occurrence of chromosomal translocations that result in fusion proteins involving FGFR’s in cancers, and it illustrates the aberrant signaling mechanisms activated by these oncogenes.

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