Recent investigations have elucidated the cytokineinduced NF-B activation pathway. IB kinase (IKK) phosphorylates inhibitors of NF-B (IBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-B. We have examined the possibility that IKK can phosphorylate the p65 NF-B subunit as well as IB in the cytokine-induced NF-B activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF-␣ in a time dependent manner similar to IB 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 IKK␤ 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-Binducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-B⅐IB complex.The transcription factor nuclear factor-B (NF-B) 1 plays a pivotal role in inflammatory and immune responses (1-3). NF-B is composed of a heterodimer of p65 and p50 subunits in most cell types and is sequestered in the cytoplasm by its inhibitor proteins, the IBs (4 -8). Several NF-B-activating agents, including pro-inflammatory cytokines, phorbol esters, and bacterial lipopolysaccaride, induce the phosphorylation of IBs at two NH 2 -terminal Ser residues. The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, thereby releasing NF-B to enter the nucleus for gene expression (9 -15).Recent investigations have focused on the phosphorylation of IBs and clearly elucidated the molecular mechanisms of the phosphorylation. In brief, two closely related kinases, designated IB kinase (IKK) ␣ and IKK␤, have been identified as components of the multiprotein IKK complex (500 -900 kDa) that directly phosphorylates the critical Ser residues of . IKK␣ and IKK␤ together form a heterodimer through their COOH-terminal leucine zipper motifs, and the functional IKK complex contains both IKK subunits. NF-B-inducing kinase (NIK), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, interacts with and activates the IKK complex (21). Other MAP3Ks, including transforming growth factor-␤ activated kinase 1 (TAK1) (22-24), MAPK/ extracellular signal-regulated kinase kinase kinases (MEKK1-3) (25-28), and Cot/Tpl2 (29), have been shown to be involved in the IKK activation pathways, indicating the important roles of MAP3K family kinases in the IKK activation by diverse extracellular stimuli.The activity of several inducible transcription factors, including cAMP response element-binding protein (CREB) (30) and c-Jun (31), has been shown to be regulated by phosphorylation. It has been shown that the p65 NF-B subun...

The NF-B 1 family of transcription factors consists of binary complexes of subunits with related promoter-binding and transactivation properties. The p65/RelA, RelB, and c-Rel subunits stimulate transcription, whereas the p50 and p52 subunits serve primarily to bind to DNA (1). The prototypical NF-B complex is the p65-p50 heterodimer (2). NF-B is sequestered in a latent form in the cytoplasm through its interaction with the inhibitory IB proteins. In response to signals, IB kinase is activated, and IB is phosphorylated and degraded, releasing NF-B, which enters the nucleus and binds to DNA (2-5). However, the phosphorylation and degradation of IB and the consequent liberation of NF-B are not sufficient to activate NF-B-dependent transcription, which also relies on a second pathway, which leads to the stimulus-induced phosphorylation of the p65/RelA, RelB, and c-Rel subunits of NF-B (6 -15).Our laboratory (13) and others (7,14) have shown that the pro-inflammatory cytokines IL-1 and TNF induce the phosphorylation and activation of the p65 subunit of NF-B, a pathway distinct from the one leading to IB degradation and NF-B nuclear translocation. Additionally, phosphatidylinositol 3Ј-kinase (PI3K) and the serine/threonine kinase AKT play critical roles in this pathway (13,16,17). Recently, an additional function for IL-1-stimulated PI3K/AKT activation has been reported: phosphorylation of the NF-B p50 subunit in response to these kinases increases the DNA-binding capacity of the NF-B complex (18).Targeted gene disruptions have demonstrated that IKK␤ (but not IKK␣) is largely responsible for cytokine-induced IB degradation and NF-B nuclear translocation (19 -24). However, IKK␣-null mouse embryo fibroblasts (MEFs) are deficient in inducing several NF-B-dependent mRNAs in response to IL-1 and TNF (21). Activated AKT interacts with IKK␣ upon cytokine stimulation and induces the phosphorylation of threonine 23 (25). These findings raise the interesting possibility that, although IKK␣ is dispensable for IB␣ degradation and NF-B nuclear translocation, it may be required in the PI3K/ AKT pathway that leads to the phosphorylation and activation of NF-B. Therefore, we have investigated the roles of the IKK ␣ and ␤ subunits in the IL-1-and TNF-mediated phosphorylation and activation of the p65 subunit of NF-B. EXPERIMENTAL PROCEDURESBiological Reagents and Cell Culture-Recombinant human IL-1␤ was from NCI, National Institutes of Health. Recombinant human TNF was from Preprotech (Rocky Hill, NJ). LY 294002 was from Sigma. Polyclonal anti-IKK␣, anti-IKK␤, anti-IKK␥, anti-p65/RelA, and anti-IB␣ antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-phospho-p38 MAPK, anti-p38 MAPK, anti-phospho-AKT, anti-AKT, anti-phospho-ERK1/2, anti-ERK1/2, anti-phosphop90 RSK , anti-p90 RSK , anti-phospho-JNK1, and anti-JNK antibodies were from Cell Signaling Technologies (Beverly, MA). Protein A-Sepharose and glutathione-agarose beads were from Amersham Biosciences, Inc. (Buckinghamshire, United Kingdom). Wild-type and...

The activation of NF-kappaB has been shown to be regulated by multiple phosphorylations of IkappaBs and the NF-kappaB 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-kappaB stimulants including TNF-alpha in the cytoplasm and rapidly dephosphorylated in the nucleus. The TNF-alpha-but not IL-1beta-induced Ser-536 phosphorylation was severely impaired in murine embryonic fibroblasts derived from traf2-/-traf5-/- mice. Bay 11-7082, an inhibitor of IkappaB phosphorylation, inhibited the TNF-alpha-induced phosphorylation in vivo. In addition, overexpression of TGF-beta-activated kinase 1 (TAK1), IKKalpha and IKKbeta stimulated the phosphorylation, and their dominant negative mutants blocked the TNF-alpha-induced phosphorylation. Moreover, small interfering RNAs (siRNAs) against TAK1, IKKalpha and IKKbeta 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 IkappaBalpha and p65, which further support the idea that both IkappaB and NF-kappaB are substrates for the IKK complex in the activation of NF-kappaB.

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The Ubc13 E2 ubiquitin-conjugating enzyme is key in the process of 'tagging' target proteins with lysine 63-linked polyubiquitin chains, which are essential for the transmission of immune receptor signals culminating in activation of the transcription factor NF-kappaB. Here we demonstrate that conditional ablation of Ubc13 resulted in defective B cell development and in impaired B cell and macrophage activation. In response to all tested stimuli except tumor necrosis factor, Ubc13-deficient cells showed almost normal NF-kappaB activation but considerably impaired activation of mitogen-activated protein kinase. Ubc13-induced activation of mitogen-activated protein kinase required, at least in part, ubiquitination of the adaptor protein IKKgamma. These results show that Ubc13 is key in the mammalian immune response.