Recent investigations have elucidated the cytokine-induced NF-κB activation pathway. IκB kinase (IKK) phosphorylates inhibitors of NF-κB (IκBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-κB. We have examined the possibility that IKK can phosphorylate the p65 NF-κB subunit as well as IκB in the cytokine-induced NF-κB activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF-α in a time dependent manner similar to IκB phosphorylation. In vitro phosphorylation with GST-fused p65 showed that a p65 phosphorylating activity was present in the cytoplasmic fraction and the target residue was Ser-536 in the carboxyl-terminal transactivation domain. The endogenous IKK complex, overexpressed IKKs, and recombinant IKKβ efficiently phosphorylated the same Ser residue of p65 in vitro. The major phosphorylation site in vivo was also Ser-536. Furthermore, activation of IKKs by NF-κB-inducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-κB·IκB complex.

The activation of NF-κB has been shown to be regulated by multiple phosphorylations of IκBs and the NF-κB p65 subunit. Here, we characterized the intracellular signaling pathway leading to phosphorylation of p65 on Ser-536 using a novel anti-phospho-p65 (Ser-536) antibody. The Ser-536 of endogenous p65 was rapidly phosphorylated in response to a wide variety of NF-κB stimulants including TNF-α in the cytoplasm and rapidly dephosphorylated in the nucleus. The TNF-α-but not IL-1β-induced Ser-536 phosphorylation was severely impaired in murine embryonic fibroblasts derived from traf2^-/-traf5^-/- mice. Bay 11-7082, an inhibitor of IκB phosphorylation, inhibited the TNF-α-induced phosphorylation in vivo. In addition, overexpression of TGF-β-activated kinase 1 (TAK1), IKKα and IKKβ stimulated the phosphorylation, and their dominant negative mutants blocked the TNF-α-induced phosphorylation. Moreover, small interfering RNAs (siRNAs) against TAK1, IKKα and IKKβ blocked the phosphorylation of endogenous p65. On the other hand, calyculin-A, a protein phosphatase inhibitor, blocked the dephosphorylation in the nucleus in vivo. These results indicate that similar signaling pathways were utilized for the phosphorylations of IκBα and p65, which further support the idea that both IκB and NF-κB are substrates for the IKK complex in the activation of NF-κB.

Phosphatidylinositol 3′-kinase (PI3K) and the serine/threonine kinase AKT have critical roles in phosphorylating and transactivating the p65 subunit of nuclear factor κB (NF-κB) in response to the pro-inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF). Mouse embryo fibroblasts (MEFs) lacking either the α or β subunit of IκB kinase (IKK) were deficient in NF-κB-dependent transcription following treatment with IL-1 or TNF. However, in contrast to IKKβ-null MEFs, IKKα-null MEFs were not substantially defective in the cytokine-stimulated degradation of Iκβα or in the nuclear translocation of NF-κB. The IKK complexes from IKKα- or IKKβ-null MEFs were both deficient in PI3K-mediated phosphorylation of the transactivation domain of the p65 subunit of NF-κB in response to IL-1 and TNF, and constitutively activated forms of PI3K or AKT did not potentiate cytokine-stimulated activation of NF-κB in either IKKα- or IKKβ-null MEFs. Collectively, these data indicate that, in contrast to IKKβ, which is required for both NF-κB liberation and p65 phosphorylation, IKKα is required solely for the cytokine-induced phosphorylation and activation of the p65 subunit of NF-κB that are mediated by the PI3K/AKT pathway.

The receptor activator of NF-κB (RANK) and its ligand RANKL are key molecules for differentiation and activation of osteoclasts. RANKL stimulates transcription factors AP-1 through mitogen-activated protein kinase (MAPK) activation, and NF-κB through IκB kinase (IKK) activation. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is essential for activation of these kinases. In the interleukin-1 signaling pathway, TAK1 MAPK kinase kinase (MAPKKK) mediates MAPK and IKK activation via interaction with TRAF6, and TAB2 acts as an adapter linking TAK1 and TRAF6. Here, we demonstrate that TAK1 and TAB2 participate in the RANK signaling pathway. Dominant negative forms of TAK1 and TAB2 inhibit NF-κB activation induced by overexpression of RANK. In 293 cells stably transfected with full-length RANK, RANKL stimulation facilitates the formation of a complex containing RANK, TRAF6, TAB2, and TAK1, leading to the activation of TAK1. Furthermore, in murine monocyte RAW 264.7 cells, dominant negative forms of TAK1 and TAB2 inhibit NF-κB activation induced by RANKL and endogenous TAK1 is activated in response to RANKL stimulation. These results suggest that the formation of the TRAF6-TAB2-TAK1 complex is involved in the RANK signaling pathway and may regulate the development and function of osteoclasts.