DNA-Dependent Protein Kinase Phosphorylation of IκBα and IκBβ Regulates NF-κB DNA Binding Properties

Regulation of the IκBα and IκBβ proteins is critical for modulating NF-κB-directed gene expression. Both IκBα and IκBβ are substrates for cellular kinases that phosphorylate the amino and carboxy termini of these proteins and regulate their function. In this study, we utilized a biochemical fractionation scheme to purify a kinase activity which phosphorylates residues in the amino and carboxy termini of both IκBα and IκBβ. Peptide microsequence analysis by capillary high-performance liquid chromatography ion trap mass spectroscopy revealed that this kinase was the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PK phosphorylates serine residue 36 but not serine residue 32 in the amino terminus of IκBα and also phosphorylates threonine residue 273 in the carboxy terminus of this protein. To determine the biological relevance of DNA-PK phosphorylation of IκBα, murine severe combined immunodeficiency (SCID) cell lines which lack the DNA-PKcs gene were analyzed. Gel retardation analysis using extract prepared from these cells demonstrated constitutive nuclear NF-κB DNA binding activity, which was not detected in extracts prepared from SCID cells complemented with the human DNA-PKcs gene. Furthermore, IκBα that was phosphorylated by DNA-PK was a more potent inhibitor of NF-κB binding than nonphosphorylated IκBα. These results suggest that DNA-PK phosphorylation of IκBα increases its interaction with NF-κB to reduce NF-κB DNA binding properties.     

Loss of IκBα-Mediated Control over Nuclear Import and DNA Binding Enables Oncogenic Activation of c-Rel

The IκBα protein is able both to inhibit nuclear import of Rel/NF-κB proteins and to mediate the export of Rel/NF-κB proteins from the nucleus. We now demonstrate that the c-Rel–IκBα complex is stably retained in the cytoplasm in the presence of leptomycin B, a specific inhibitor of Crm1-mediated nuclear export. In contrast, leptomycin B treatment results in the rapid and complete relocalization of the v-Rel–IκBα complex from the cytoplasm to the nucleus. IκBα also mediates the rapid nuclear shuttling of v-Rel in an interspecies heterokaryon assay. Thus, continuous nuclear export is required for cytoplasmic retention of the v-Rel–IκBα complex. Furthermore, although IκBα is able to mask the c-Rel-derived nuclear localization sequence (NLS), IκBα is unable to mask the v-Rel-derived NLS in the context of the v-Rel–IκBα complex. Taken together, our results demonstrate that IκBα is unable to inhibit nuclear import of v-Rel. We have identified two amino acid differences between c-Rel and v-Rel (Y286S and L302P) which link the failure of IκBα to inhibit nuclear import and DNA binding of a mutant c-Rel protein to oncogenesis. Our results support a model in which loss of IκBα-mediated control over c-Rel leads to oncogenic activation of c-Rel.     

TRIM22 activates NF-κB signaling in glioblastoma by accelerating the degradation of IκBα

NF-κB signaling plays a critical role in tumor growth and treatment resistance in GBM as in many other cancers. However, the molecular mechanisms underlying high, constitutive NF-κB activity in GBM remains to be elucidated. Here, we screened a panel of tripartite motif (TRIM) family proteins and identified TRIM22 as a potential activator of NF-κB using an NF-κB driven luciferase reporter construct in GBM cell lines. Knockout of TRIM22 using Cas9-sgRNAs led to reduced GBM cell proliferation, while TRIM22 overexpression enhanced proliferation of cell populations, in vitro and in an orthotopic xenograft model. However, two TRIM22 mutants, one with a critical RING-finger domain deletion and the other with amino acid changes at two active sites of RING E3 ligase (C15/18A), were both unable to promote GBM cell proliferation over controls, thus implicating E3 ligase activity in the growth-promoting properties of TRIM22. Co-immunoprecipitations demonstrated that TRIM22 bound a negative regulator of NF-κB, NF-κB inhibitor alpha (IκBα), and accelerated its degradation by inducing K48-linked ubiquitination. TRIM22 also formed a complex with the NF-κB upstream regulator IKKγ and promoted K63-linked ubiquitination, which led to the phosphorylation of both IKKα/β and IκBα. Expression of a non-phosphorylation mutant, srIκBα, inhibited the growth-promoting properties of TRIM22 in GBM cell lines. Finally, TRIM22 was increased in a cohort of primary GBM samples on a tissue microarray, and high expression of TRIM22 correlated with other clinical parameters associated with progressive gliomas, such as wild-type IDH1 status. In summary, our study revealed that TRIM22 activated NF-κB signaling through posttranslational modification of two critical regulators of NF-κB signaling in GBM cells.Subject terms: CNS cancer, Oncogenes, Ubiquitin ligases     

Postrepression Activation of NF-κB Requires the Amino-Terminal Nuclear Export Signal Specific to IκBα

One of the most prominent NF-kappaB target genes in mammalian cells is the gene encoding one of its inhibitor proteins, IkappaBalpha. The increased synthesis of IkappaBalpha leads to postinduction repression of nuclear NF-kappaB activity. However, it is unknown why IkappaBalpha, among multiple IkappaB family members, is involved in this process and what significance this feedback regulation has beyond terminating NF-kappaB activity. Herein, we report an important IkappaBalpha-specific function dictated by its amino-terminal nuclear export sequence (N-NES). The IkappaBalpha N-NES is necessary for the postinduction export of nuclear NF-kappaB, which is a critical event in reestablishing a permissive condition for NF-kappaB to be rapidly reactivated. We show that although IkappaBalpha and another IkappaB member, IkappaBbeta, can enter the nucleus and repress NF-kappaB DNA-binding activity during the postinduction phase, only IkappaBalpha allows the efficient export of nuclear NF-kappaB. Moreover, swapping the N-terminal region of IkappaBbeta for the corresponding IkappaBalpha sequence is sufficient for the IkappaB chimera protein to export NF-kappaB similarly to IkappaBalpha during the postinduction state. Our findings provide a mechanistic explanation of why IkappaBalpha but not other IkappaB members is crucial for postrepression activation of NF-kappaB. We propose that this IkappaBalpha-specific function is important for certain physiological and pathological conditions where NF-kappaB needs to be rapidly reactivated.     

Cell-Specific Association and Shuttling of IκBα Provides a Mechanism for Nuclear NF-κB in B Lymphocytes

Mature B lymphocytes are unique in containing nuclear Rel proteins prior to cell stimulation. This activity consists largely of p50-c-Rel heterodimers, and its importance for B-cell function is exemplified by reduced B-cell viability in several genetically altered mouse strains. Here we suggest a mechanism for the cell specificity and the subunit composition of constitutive B-cell NF-kappaB based on the observed properties of Rel homo- and heterodimers and IkappaBalpha. We show that c-Rel lacks a nuclear export sequence, making the removal of c-Rel-containing complexes from the nucleus less efficient than removal of p65-containing complexes. Second, the nuclear import potential of p65 and c-Rel homodimers but not p50-associated heterodimers was attenuated when they were complexed to IkappaBalpha, leading to a greater propensity of heterodimers to be nuclear. We propose that subunit composition of B-cell NF-kappaB reflects the inefficient retrieval of p50-c-Rel heterodimers from the nucleus. Cell specificity may be a consequence of c-Rel-IkappaBalpha complexes being present only in mature B cells, which leads to nuclear c-Rel due to IkappaBalpha turnover and shuttling of the complex.     

Dynamic chromatin association of IκBα is regulated by acetylation and cleavage of histone H4

Nuclear IκBα preferentially binds the acetylated N‐terminal tail of histone H4 in vivo, specifically in the skin and intestine stem cell compartments. N‐terminal cleavage of histone H4 facilitates IκBα dissociation and cellular differentiation.     

Roles of IκB kinase ε in the innate immune defense and beyond

IκB kinase ε (IKKε) is a non-canonical IκB kinase that is extensively studied in the context of innate immune response. Recently, significant progress has been made in understanding the role of IKKε in interferon (IFN) signaling. In addition to its roles in innate immunity, recent studies also demonstrate that IKKε is a key regulator of the adaptive immune response. Specifically, IKKε functions as a negative feedback kinase to curtail CD8 T cell response, implying that it can be a potential therapeutic target to boost antiviral and antitumor T cell immunity. In this review, we highlight the roles of IKKε in regulating IFN signaling and T cell immunity, and discuss a few imminent questions that remain to be answered.