Although the posttranslational stability of NIK is the central regulatory element of the noncanonical NF-κB pathway, the molecular mechanism by which the degradation of NIK is controlled in different contexts is only just beginning to be understood (8
). Constitutive degradation of NIK in unstimulated cells is mediated by the TRAF-cIAP complex; however, little is known about control of NIK after receptor stimulation (12
). In this report, we describe a previously uncharacterized negative feedback mechanism within noncanonical signaling that controls the abundance of NIK after receptor ligation. Negative feedback occurred through IKKα-mediated phosphorylation of serine residues in the C terminus of NIK that promoted its destabilization. Consequently, receptor-induced stabilization of NIK and the resultant activation of the kinase activity of IKKα ultimately fed back to prevent the uncontrolled accumulation of NIK and enhanced noncanonical NF-κB signaling after receptor stimulation. IKKα has traditionally been viewed as a downstream target of the kinase activity of NIK within noncanonical NF-κB signaling, a component of the pathway that is activated by NIK and that transmits that signal forward through the phosphorylation of p100 (9
). However, our study demonstrates that IKKα plays a critical role in the regulation of noncanonical NF-κB signaling by additionally acting as an upstream kinase of NIK and in so doing, controlling the stability of NIK ().
How the stability of NIK is controlled and the effect of its dysregulation in vivo have been of substantial interest. To date, all such studies have focused on the roles of TRAF2 and TRAF3 in controlling the degradation of NIK. For example, in the past several years various groups have generated mice that lack TRAF2
in combination with p100
, that have conditional deletion of TRAF2
in various cell types, and that transgenically express NIK mutants that cannot interact with TRAF3 (13
). We addressed the possibility that IKKα-mediated phosphorylation of NIK might somehow be required for activity of the TRAF-cIAP complex; however, our studies suggested that this was not the case because the abundance of NIK in IKK
cells and aly
cells continued to be responsive to stimuli that modulated the activity of the TRAF-cIAP complex. Furthermore, the affinity of NIK for TRAF3, the only component of the TRAF-cIAP complex thought to interact with NIK, did not change in the presence or absence of IKKα and was not affected by inclusion of the 3SA mutation in NIK. These data are in accordance with findings that the serine residues in the C terminus of NIK that are phosphorylated by IKKα are far from the N-terminal TRAF3-binding motif of NIK. Finally, the degradation of NIK in cells in which all of the components of the TRAF-cIAP complex were ectopically coexpressed was not affected by the presence of the 3SA mutation. Therefore, it appears that the abundance of NIK is controlled by two separate mechanisms: the previously studied TRAF-cIAP complex, which is responsible for the degradation of NIK in unstimulated contexts, and the previously uncharacterized IKKα-dependent mechanism that we describe here, which is responsible for the inhibition of NIK after receptor stimulation.
That the differential stabilities of reconstituted wild-type NIK and NIK-3SA were eliminated upon inhibition of the proteasome suggests that proteasomal degradation may be the ultimate mechanism by which feedback phosphorylation of NIK results in its degradation (). Proteins degraded by the proteasome are frequently targeted for destruction by ubiquitination (32
). However, we were unable to detect any enhancement in the extent of ubiquitination of NIK in HEK 293T cells cotransfected with plasmids encoding IKKα and NIK (fig. S5
). Although ubiquitination-independent degradation by the proteasome has been described for a number of proteins, it is also possible that the necessary endogenous cofactors required for mediating phosphorylation-dependent ubiquitination of NIK are not found in sufficient abundance in HEK 293T cells to exert detectable ubiquitination of NIK (33
). If ubiquitination is ultimately found to be the molecular mechanism by which phosphorylated NIK is degraded, it will be of interest to determine whether the lysine residues in NIK that are targeted by ubiquitin are different from those targeted by the TRAF-cIAP complex.
In the case of p100, whose ubiquitination and proteasomal processing is stimulated by IKKα-mediated phosphorylation, ubiquitination is mediated by recruitment of the SCFβ-TrCP
ubiquitin ligase complex (10
). We investigated whether a similar mechanism might be operational for NIK, but we found no enhancement in the recruitment of β-TrCP to NIK in comparison to that to NIK-3SA in the presence or absence of IKKα. Furthermore, the phosphorylation site that we identified bears no homology to the well-described β-TrCP consensus phosphodegron (34
). Further studies will be required to elaborate the molecular constituents that stimulate the degradation of NIK in response to IKKα-dependent phosphorylation of the C-terminal serine residues that we identified.
In fibroblasts derived from IKKα-deficient mice or aly mice, the basal stability of NIK in the absence of stimulation was also increased, to an extent comparable to that seen in fibroblasts deficient in components of the TRAF-cIAP complex. Therefore, it is possible that, for some cell types, both the TRAF-cIAP complex and the IKKα-dependent phosphorylation system are nonredundantly required to completely eliminate all of the NIK protein that is synthesized during the unstimulated state. This is consistent with the observation that IKKα-mediated phosphorylation of NIK in fibroblasts appeared to occur even in the absence of receptor ligation, as evidenced by the difference in the apparent molecular mass of NIK species from IKKα+/+ and IKKα−/− fibroblasts even under conditions in which just the proteasome was inhibited () and by the observation of the phosphorylation of NIK by endogenous IKKα in unstimulated fibroblasts ().
In addition to its role in noncanonical NF-κB signaling, NIK can enhance canonical NF-κB signaling in certain cellular contexts through the direct activation of the IKK complex (35
). For example, NIK stabilized in fibroblasts because of a deficiency in TRAF3 or because of pretreatment with agonists of the LTβR results in the substantial enhancement of TNFα-induced activation of the IKK complex (35
). Activation of canonical NF-κB signaling by NIK is dependent on functional IKKα (35
). Consequently, it may be that in the absence of IKKα-dependent destabilization of NIK, canonical NF-κB signaling may also be enhanced.
Enhanced noncanonical signaling is now recognized to cause various hematological cancers and autoimmune pathology. Thus far, this is thought to occur through deletion of components of the TRAF-cIAP complex, genetic amplifications of NIK
, or long-term ligand stimulation of cells by increased concentrations of BAFF in the plasma (14
). Notably, defective negative feedback in canonical NF-κB signaling leads to malignancies (24
). As such, determining the in vivo sequelae of defective negative feedback of noncanonical NF-κB signaling in various cell types will be of important interest for future studies.