Initiation of the genetic programs for inflammation and immunity involves nuclear mobilization of transcription factor NF-B. This signal-dependent process is controlled in part by the ␤-catalytic subunit of IB kinase (IKK␤), which marks IB␣ and other cytoplasmic inhibitors of NF-B for proteolytic destruction. The catalytic activity of IKK␤ is stimulated by pathologic and physiologic inducers of NF-B, such as the Tax oncoprotein and proinflammatory cytokines. We now report evidence that these NF-B inducers target IKK␤ for conjugation to ubiquitin (Ub) in mammalian cells. The apparent molecular size of modified IKK␤ is compatible with monoubiquitination rather than attachment of a multimeric Ub chain. The modification is contingent upon signal-induced phosphorylation of the activation T loop in IKK␤ at Ser-177/Ser-181. The formation of IKK␤-Ub conjugates is disrupted in cells expressing YopJ, a Ub-like protein protease that interferes with the NF-B signaling pathway. These findings indicate an important mechanistic link between phosphorylation, ubiquitination, and the biologic action of IKK␤.The inducible transcription factor NF-B is biochemically coupled to cell-surface members of the tumor necrosis factor (TNF) 1 receptor, Toll-like receptor, and immunoglobulin superfamilies (1). NF-B is persistently activated in cells expressing the Tax protein of human T-cell leukemia virus type 1, which has potent oncogenic properties (2). This deregulated pattern of NF-B activity also underlies acute and chronic inflammatory diseases (1). Nuclear translocation of NF-B is controlled by an inducible multicomponent protein kinase, termed IKK, which targets IB␣ and other cytoplasmic inhibitors of NF-B for proteolytic destruction (3). IKK contains two catalytic subunits, designated IKK␣ and IKK␤, as well as a regulatory subunit called IKK␥ (3). IKK␤ and IKK␥ are essential for proteolytic inactivation of IB␣, whereas IKK␣ mediates NF-B subunit processing via an IKK␥-independent mechanism (4).The catalytic activity of IKK␤ is stimulated by signals that trigger its phosphorylation at Ser-177/Ser-181, such as TNF-␣ (5). These phosphoacceptors lie in a region of the catalytic domain that shares homology with regulatory "T loop" sequences found in mitogen-activated protein kinases and their upstream activators (5). In contrast to its transient pattern of phosphorylation and activation in TNF-stimulated cells, IKK␤ is chronically phosphorylated and activated in cells expressing the Tax oncoprotein (6). Tax-induced activation of IKK␤ is blocked by YopJ (6), a cysteine protease that removes ubiquitin (Ub)-related modifiers from target proteins in mammalian cells (7). This finding with YopJ raised the possibility that IKK␤ might be subject to signal-dependent ubiquitination. In keeping with this possibility, previous studies with partially purified kinase complexes suggested that IKK is conjugated to Ub in vitro (8). However, these pioneering in vitro experiments were conducted prior to molecular cloning of individual IKK subunits (3).We n...

Transcription factor NF-B and other dimeric members of the Rel polypeptide family regulate the expression of multiple genes involved in inflammation, immunity, mitosis, and cell survival (1-3). Biologic inducers of NF-B include the proinflammatory cytokines tumor necrosis factor ␣ (TNF) 1 and interleukin-1 (IL-1), the lipopolysaccharide (LPS) component of Gram-negative bacteria, and the Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV1) (4 -7). Each of these signal-dependent responses is controlled by labile cytoplasmic inhibitors of NF-B such as IB␣ and a multicomponent IB kinase called IKK (8). The core IKK holoenzyme contains two catalytic subunits termed IKK␣ and IKK␤. Following cellular stimulation, the IKK␤ catalytic subunit phosphorylates IB␣, leading to degradation of the inhibitor and nuclear translocation of NF-B. Signal-dependent activation of IKK␤ is triggered by phosphorylation of two serine residues in its "T loop" regulatory domain (9). This modification appears to involve either autophosphorylation or phosphoryl group transfer from an upstream IKK␤ kinase to the same acceptor sites following cellular stimulation (9).More recent studies of the IB kinase complex have identified a noncatalytic component called IKK␥ (also known as NEMO, IKKAP1, or FIP-3) (10 -13). IKK␥ is required for signaldependent activation of IKK␤, leading to its assignment as an essential regulatory subunit of the enzyme (10,12,14). Small deletions or point mutations in the gene encoding IKK␥ can cause skin inflammation or humoral immunodeficiencies in humans (15). In terms of structural organization, sequences within the NH 2 -terminal half of IKK␥ mediate its interaction with IKK␤ (11). In contrast, the COOH-terminal half of IKK␥ is required for signal-dependent regulation of IB kinase activity, suggesting that IKK␥ links IKK␤ to upstream activators (11,12). Despite all of these findings, the mechanism of IKK␥ action remains elusive (16). In this regard, we and others (9,(17)(18)(19) have recently shown that IKK␥ is phosphorylated in response to NF-B agonists such as TNF and the Tax oncoprotein of HTLV1. Considering the key role that phosphorylation plays in the mechanism for IKK␤ activation, these findings suggest that IKK␥ subunit phosphorylation is important for proper regulation of the NF-B signaling pathway.To extend these fundamental observations, we conducted new experiments that address the mechanism of IKK␥ phosphorylation and the relevant phosphoacceptor sites. In this report, we demonstrate that endogenous IKK␤ but not IKK␣ is required for signal-dependent phosphorylation of IKK␥ in vivo. Using a combination of site-directed mutagenesis and phosphopeptide mapping, we have also monitored changes in the phosphorylation status of IKK␥ in metabolically radiolabeled cells. Results from these biochemical experiments indicate that human IKK␥ is phosphorylated at Ser-31, Ser-43, and Ser-376 in response to cellular stimulation with either TNF or the HTLV1 Tax oncoprotein. Minimal deletion of the zinc finger domai...