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Transcription factor NF-B plays a key regulatory role in the cellular response to pro-inflammatory cytokines such as tumor necrosis factor-␣ (TNF). In the absence of TNF, NF-B is sequestered in the cytoplasm by inhibitory IB proteins. Phosphorylation of IB by the ␤-catalytic subunit of IKK, a multicomponent IB kinase, targets the inhibitor for proteolytic destruction and facilitates nuclear translocation of NF-B. This pathway is initiated by TNF-dependent phosphorylation of T loop serines in IKK␤, which greatly stimulates IB kinase activity. Prior in vitro mixing experiments indicate that protein serine/threonine phosphatase 2A (PP2A) can dephosphorylate these T loop serines and inactivate IKK, suggesting a negative regulatory role for PP2A in IKK signaling. Here we provided several in vivo lines of evidence indicating that PP2A plays a positive rather than a negative role in the regulation of IKK. First, TNF-induced degradation of IB is attenuated in cells treated with okadaic acid or fostriecin, two potent inhibitors of PP2A. Second, PP2A forms stable complexes with IKK in untransfected mammalian cells. This interaction is critically dependent on amino acid residues 121-179 of the IKK␥ regulatory subunit. Third, deletion of the PP2A-binding site in IKK␥ attenuates T loop phosphorylation and catalytic activation of IKK␤ in cells treated with TNF. Taken together, these data provide strong evidence that the formation of IKK⅐PP2A complexes is required for the proper induction of IB kinase activity in vivo.Insults to the immune system and various forms of cellular stress activate the transcription factor NF-B 3 and other dimeric members of the Rel polypeptide family (reviewed in Refs. 1 and 2). NF-B regulates the expression of multiple genes involved in the control of cell growth, division, and survival. A variety of stimuli can activate NF-B-mediated gene transcription, including tumor necrosis factor-␣ (TNF), interleukin-1, T and B cell mitogens, bacterial products, viral proteins, doublestranded RNA, as well as physical and chemical stress. Most of these agonists converge on a latent, cytoplasmic form of NF-B that associates with IB␣ or other members of the inhibitory IB family. Following cellular stimulation, IB␣ is phosphorylated, ubiquitinated, and degraded by the 26 S proteasome. In turn, NF-B is free to translocate to the nuclear compartment where it activates transcription units containing the B-binding site (1, 2).Phosphorylation of IB is catalyzed by a multicomponent protein kinase termed the IKK signalsome (3). Within the prototypic complex are two highly homologous IB kinase subunits, termed IKK␣ and IKK␤, which form homo-or heterodimers via their leucine zippers (4 -6). In response to various cell stimuli, IKK␣ and IKK␤ are activated by a mechanism involving phosphorylation of their respective T loops at Ser-176/Ser-180 and Ser-177/Ser-181 (7). T loop phosphorylation occurs via IKK subunit trans-autophosphorylation and/or the action of upstream kinases (3). In addition to phosphorylation of T loop ser...

The Tax transforming protein encoded by human Tcell leukemia virus type 1 (HTLV1) persistently activates transcription factor NF-B and deregulates the expression of downstream genes that mediate cell cycle entry. We recently found that Tax binds to and chronically stimulates the catalytic function of IB kinase (IKK), a cellular enzyme complex that phosphorylates and inactivates the IB inhibitory subunit of NF-B. We now demonstrate that the IKK␤ catalytic subunit and IKK␥ regulatory subunit of IKK are chronically phosphorylated in HTLV1-infected and Tax-transfected cells. Alanine substitutions at Ser-177 and Ser-181 in the T loop of IKK␤ protect both of these IKK subunits from Tax-directed phosphorylation and prevent the induction of IB kinase activity. Each of these inhibitory effects is recapitulated in Tax transfectants expressing the bacterial protein YopJ, a potent in vivo agonist of T loop phosphorylation. Moreover, ectopically expressed forms of IKK␤ that contain glutamic acid substitutions at Ser-177 and Ser-181 have the capacity to phosphorylate a recombinant IKK␥ substrate in vitro. We conclude that Tax-induced phosphorylation of IKK␤ is required for IKK␤ activation, phosphoryl group transfer to IKK␥, and acquisition of the deregulated IKK phenotype.

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 governs the expression of multiple genes involved in cell growth, immunity, and inflammation. Nuclear translocation of NF-B is regulated from the cytoplasm by IB kinase-␤ (IKK␤), which earmarks inhibitors of NF-B for polyubiquination and proteasome-mediated degradation. Activation of IKK␤ is contingent upon signal-induced phosphorylation of its T loop at Ser-177/Ser-181. T loop phosphorylation also renders IKK␤ a substrate for monoubiquitination in cells exposed to chronic activating cues, such as the Tax oncoprotein or sustained signaling through proinflammatory cytokine receptors. Here we provide evidence that the T loop-proximal residue Lys-163 in IKK␤ serves as a major site for signal-induced monoubiquitination with significant regulatory potential. Conservative replacement of Lys-163 with Arg yielded a monoubiquitination-defective mutant of IKK␤ that retains kinase activity in Tax-expressing cells but is impaired for activation mediated by chronic signaling from the type 1 receptor for tumor necrosis factor-␣. Phosphopeptide mapping experiments revealed that the Lys-163 3 Arg mutation also interferes with proper in vivo but not in vitro phosphorylation of cytokine-responsive serine residues located in the distal C-terminal region of IKK␤. Taken together, these data indicate that chronic phosphorylation of IKK␤ at Ser-177/Ser-181 leads to monoubiquitin attachment at nearby Lys-163, which in turn modulates the phosphorylation status of IKK␤ at select C-terminal serines. This mechanism for post-translational cross-talk may play an important role in the control of IKK␤ signaling during chronic inflammation.

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...

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