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An IkappaB kinase (IKK) signalsome that phosphorylates IkappaB targeting IkappaB for degradation

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is controlled by sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit IkappaB. A large multiprotein complex, the IkappaB kinase (IKK) signalsome, has been purified from HeLa cells and has been found to contain a cytokine-inducible IkappaB kinase activity that phosphorylates IkappaB-alpha and IkappaB-beta. Two components of the IKK signalsome, IKK-1 and IKK-2, are closely related protein serine kinases containing leucine zipper and helix-loop-helix protein interaction motifs. Mutant versions of IKK-2 have pronounced effects on RelA nuclear translocation and NF-kappaB-dependent reporter activity, consistent with a critical role for the IKK kinases in the NF-kappaB signaling pathway (Mercurio, 1997).

Activation of the transcription factor NF-kappaB is controlled by the sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit, IkappaB. A large multiprotein complex, the IkappaB kinase (IKK) signalsome, has been isolated that contains two regulated IkappaB kinases, IKK1 and IKK2, that can each phosphorylate IkappaBalpha and IkappaBbeta. The IKK signalsome contains several additional proteins presumably required for the regulation of the NFkappaB signal transduction cascade in vivo. IkappaB kinase activity can be reassembled in vitro by using purified recombinant IKK1 and IKK2. Recombinant IKK1 or IKK2 forms homo- or heterodimers, suggesting the possibility that similar IKK complexes exist in vivo. Indeed, in HeLa cells two distinct IKK complexes, one containing IKK1-IKK2 heterodimers and the other containing IKK2 homodimers, have been identified that display differing levels of activation following tumor necrosis factor alpha stimulation. To better elucidate the nature of the IKK signalsome, an attempt has been made to identify IKK-associated proteins. To this end, a novel component common to both complexes, named IKK-associated protein 1 (IKKAP1) has been purified and cloned. In vitro, IKKAP1 associates specifically with IKK2 but not IKK1. Functional analyses reveal that binding to IKK2 requires sequences contained within the N-terminal domain of IKKAP1. Mutant versions of IKKAP1, which either lack the N-terminal IKK2-binding domain or contain only the IKK2-binding domain, disrupt the NF-kappaB signal transduction pathway. IKKAP1 therefore appears to mediate an essential step of the NF-kappaB signal transduction cascade. Heterogeneity of IKK complexes in vivo may provide a mechanism for differential regulation of NF-kappaB activation (Mercurio, 1999).

IkappaB is a target of phosphorylation, which targets the IkappaB protein for degradation resulting in the subsequent activation of NFkappaB and NFkappaB's transit into the nucleus. A 900 kDa cytokine-responsive IkappaB kinase complex (IKK) has been purified and one of its subunits, IKKalpha, a serine kinase has been molecularly cloned. IKKalpha is a NIK interacting protein. NIK was originally isolated through interaction with TRAF2, a component of the TNF receptor signaling complex, which plays a critical role in NF-kappaB activation. NIK is thought to directly interact with IKKalpha, phosphorylating and activating it. IKKbeta, a second subunit of the IKK complex, is 50% identical to IKKalpha and like it contains a kinase domain, a leucine zipper, and a helix-loop-helix. Although IKKalpha and IKKbeta can undergo homotypic interaction, they also interact with each other, and the functional IKK complex contains both subunits. The catalytic activities of both IKKalpha and IKKbeta make essential contributions to IkappaB phosphorylation and NF-kappaB activation. While the interactions between IKKalpha and IKKbeta may be mediated through their leucine zipper motifs, their helix-loop-helix motifs may be involved in interactions with essential regulatory subunits (Zandi, 1997).

Interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) stimulate transcription factors AP-1 and NF-kappaB through activation of the MAP kinases (JNK and p38) and the IkappaB kinase (IKK), respectively. The TNF-alpha and IL-1 signals are transduced through TRAF2 and TRAF6, respectively. Overexpressed TRAF2 or TRAF6 activate JNK, p38, or IKK in the absence of extracellular stimulation. By replacing the carboxy-terminal TRAF domain of TRAF2 and TRAF6 with repeats of the immunophilin FKBP12, it has been demonstrated that their effector domains are composed of their amino-terminal Zn and RING fingers. Oligomerization of the TRAF2 effector domain results in specific binding to MEKK1, a protein kinase capable of JNK, p38, and IKK activation, and induction of TNF-alpha and IL-1 responsive genes. TNF-alpha also enhances the binding of native TRAF2 to MEKK1 and stimulates the kinase activity of the latter. Thus, TNF-alpha and IL-1 signaling is based on oligomerization of TRAF2 and TRAF6 leading to activation of effector kinases (Baud, 1999).

The transcription factor NF-kappaB coordinates the activation of numerous genes in response to pathogens and pro-inflammatory cytokines, and is, therefore, vital in the development of acute and chronic inflammatory diseases. NF-kappaB is activated by phosphorylation of its inhibitory subunit, IkappaB-alpha, on serine residues 32 and 36 by cytokine-activated IKappaB kinases (IKKs); this phosphorylation precedes rapid degradation of IkappaB. IKK-alpha and IKK-beta isozymes are found in large complexes of relative molecular mass 700,000-900,000 [M(r) 70K-90K], but little is known about other components that organize and regulate these complexes. IKK-alpha was independently discovered as a NF-kappaB-inducing kinase (NIK)-associated protein in a yeast two-hybrid screen, and IKK-beta was also identified by homology screening. It is, however, unknown whether NIK is part of the IKK complex. Large, interleukin-1-inducible IKK complexes have been isolated that contain NIK, IKK-alpha, IKK-beta, IkappaB-alpha, NF-kappaB/RelA and a protein of M(r) 150K. This latter component is a new protein, termed IKK-complex-associated protein (IKAP), which can bind NIK and IKKs and assemble them into an active kinase complex. IKAP is a scaffold protein and a regulator for three different kinases involved in pro-inflammatory cytokine signaling (Cohen, 1998).

Pro-inflammatory cytokines activate the transcription factor NF-kappaB by stimulating the activity of a protein kinase that phosphorylates IkappaB, an inhibitor of NF-kappaB, at sites that trigger its ubiquitination and degradation. This results in the nuclear translocation of freed NF-kappaB dimers and the activation of transcription of target genes. Many of these target genes code for immunoregulatory proteins. A large, cytokine-responsive IkappaB kinase (IKK) complex has been purified and the genes encoding two of its subunits have been cloned. These subunits, IKK-alpha and IKK-beta, are protein kinases whose function is needed for NF-kappaB activation by pro-inflammatory stimuli. Using a monoclonal antibody against IKK-alpha, the IKK complex has been purified to homogeneity from human cell lines. IKK is found to be composed of similar amounts of IKK-alpha, IKK-beta and two other polypeptides, for which partial sequences have been obtained. These polypeptides are differentially processed forms of a third subunit, IKK-gamma. Molecular cloning and sequencing indicate that IKK-gamma is composed of several potential coiled-coil motifs. IKK-gamma interacts preferentially with IKK-beta and is required for the activation of the IKK complex. An IKK-gamma carboxy-terminal truncation mutant that still binds IKK-beta blocks the activation of IKK and NF-kappaB (Rothwarf, 1998).

A critical step in the signal-induced activation of the transcription factor NF-kappaB is the site-specific phosphorylation of its inhibitor, IkappaB, that targets the latter for degradation by the ubiquitin-proteasome pathway. Mitogen-activated protein kinase/ERK kinase kinase 1 (MEKK1) can induce both this site-specific phosphorylation of IkappaBalpha at Ser-32 and Ser-36 in vivo and the activity of a high molecular weight IkappaB kinase complex in vitro. Subsequently, others have identified two proteins, IkappaB kinase alpha (IKK-alpha) and IkappaB kinase beta (IKK-beta), that are present in a tumor necrosis factor alpha-inducible, high molecular weight IkappaB kinase complex. These kinases are believed to directly phosphorylate IkappaB based on the examination of the kinase activities of IKK immunoprecipitates, but more rigorous proof of this has yet to be demonstrated. Recombinant IKK-alpha and IKK-beta can, in fact, directly phosphorylate IkappaBalpha at Ser-32 and Ser-36, as well as homologous residues in IkappaBbeta in vitro, and thus are bona fide IkappaB kinases. MEKK1 can induce the activation of both IKK-alpha and IKK-beta in vivo. IKK-alpha is present in the MEKK1-inducible, high molecular weight IkappaB kinase complex and treatment of this complex with MEKK1 induces phosphorylation of IKK-alpha in vitro. It is concluded that IKK-alpha and IKK-beta can mediate the NF-kappaB-inducing activity of MEKK1 (Lee, 1998).

NF-kappaB is activated by various stimuli, including inflammatory cytokines and stresses. A key step in the activation of NF-kappaB is the phosphorylation of its inhibitors, IkappaBs, by an IkappaB kinase (IKK) complex. Recently, two closely related kinases, designated IKKalpha and IKKbeta, have been identified as the components of the IKK complex that phosphorylate critical serine residues of IkappaBs for degradation. A previously identified NF-kappaB-inducing kinase (NIK), which mediates NF-kappaB activation by TNFalpha and IL-1, has been demonstrated to activate IKK. Previous studies have shown that mitogen-activated protein kinase/ERK kinase kinase-1 (MEKK1), which constitutes the c-Jun N-terminal kinase/stress-activated protein kinase pathway, also activates NF-kappaB by an undefined mechanism. Overexpression of MEKK1 preferentially stimulates the kinase activity of IKKbeta, which results in phosphorylation of IkappaBs. Moreover, a catalytically inactive mutant of IKKbeta blocks the MEKK1-induced NF-kappaB activation. By contrast, overexpression of NIK stimulates kinase activities of both IKKalpha and IKKbeta comparably, suggesting a qualitative difference between NIK- and MEKK1-mediated NF-kappaB activation pathways. Collectively, these results indicate that NIK and MEKK1 independently activate the IKK complex and that the kinase activities of IKKalpha and IKKbeta are differentially regulated by two upstream kinases, NIK and MEKK1, which are responsive to distinct stimuli (Nakano, 1998).

Stimulation of cells with various proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), induces nuclear NF-kappaB expression. TNF-alpha signaling involves the recruitment of at least three proteins (TRADD, RIP, and TRAF2) to the type 1 TNF-alpha receptor tail, leading to the sequential activation of the downstream NF-kappaB-inducing kinase (NIK) and IkappaB-specific kinases (IKKalpha and IKKbeta). When activated, IKKalpha and IKKbeta directly phosphorylate the two N-terminal regulatory serines within IkappaBalpha, triggering ubiquitination and rapid degradation of this inhibitor in the 26S proteasome. This process liberates the NF-kappaB complex, allowing it to translocate to the nucleus. In studies of NIK, it was found that Thr-559 located within the activation loop of its kinase domain regulates NIK action. Alanine substitution of Thr-559 but not other serine or threonine residues within the activation loop abolishes its activity and its ability to phosphorylate and activate IKKalpha. Such a NIK-T559A mutant also dominantly interferes with TNF-alpha induction of NF-kappaB. Ectopically expressed NIK spontaneously forms oligomers and also displays a high level of constitutive activity. Analysis of a series of NIK deletion mutants indicates that multiple subregions of the kinase participate in the formation of these NIK-NIK oligomers. NIK also physically assembles with downstream IKKalpha; however, this interaction is mediated through a discrete C-terminal domain within NIK located between amino acids 735 and 947. When expressed alone, this C-terminal NIK fragment functions as a potent inhibitor of TNF-alpha-mediated induction of NF-kappaB and alone is sufficient to disrupt the physical association of NIK and IKKalpha. Together, these findings provide new insights into the molecular basis for TNF-alpha signaling, suggesting an important role for heterotypic and possibly homotypic interactions of NIK in this response (Lin, 1998).

NF-kappaB comprises a family of cellular transcription factors that are involved in the inducible expression of a variety of cellular genes that regulate the inflammatory response. NF-kappaB is sequestered in the cytoplasm by inhibitory proteins [e.g., I(kappa)B], which are phosphorylated by a cellular kinase complex known as IKK. IKK is made up of two kinases, IKK-alpha and IKK-beta, which phosphorylate I(kappa)B, leading to its degradation and translocation of NF-kappaB to the nucleus. IKK kinase activity is stimulated when cells are exposed to the cytokine TNF-alpha or by overexpression of the cellular kinases MEKK1 and NIK. The anti-inflammatory agents aspirin and sodium salicylate specifically inhibit IKK-beta activity in vitro and in vivo. The mechanism of aspirin and sodium salicylate inhibition is due to the binding of these agents to IKK-beta to reduce ATP binding. These results indicate that the anti-inflammatory properties of aspirin and salicylate are mediated in part by their specific inhibition of IKK-beta, thereby preventing activation by NF-kappaB of genes involved in the pathogenesis of the inflammatory response (Yin, 1998).

IkappaB kinases (IKKalpha and IKKbeta) are key components of the IKK complex that mediates activation of the transcription factor NF-kappaB in response to extracellular stimuli such as inflammatory cytokines, viral and bacterial infection, and UV irradiation. Although NF-kappaB-inducing kinase (NIK) interacts with and activates the IKKs, the upstream kinases for the IKKs still remain obscure. Mitogen-activated protein kinase kinase 1 (MEKK1) has been identified as an immediate upstream kinase of the IKK complex. MEKK1 is activated by tumor necrosis factor alpha (TNF-alpha) and interleukin-1 and can potentiate the stimulatory effect of TNF-alpha on IKK and NF-kappaB activation. In contrast, the dominant negative mutant of MEKK1 partially blocks activation of IKK by TNF-alpha. MEKK1 interacts with and stimulates the activities of both IKKalpha and IKKbeta in transfected HeLa and COS-1 cells and directly phosphorylates the IKKs in vitro. Furthermore, MEKK1 appears to act in parallel to NIK, leading to synergistic activation of the IKK complex. The formation of the MEKK1-IKK complex versus the NIK-IKK complex may provide a molecular basis for regulation of the IKK complex by various extracellular signals (Nemoto, 1998).

The atypical protein kinase C (PKC) isotypes (lambda/iotaPKC and zetaPKC) have been shown to be critically involved in important cell functions, such as proliferation and survival. Previous studies have demonstrated that the atypical PKCs are stimulated by tumor necrosis factor alpha (TNF-alpha) and are required for the activation of NF-kappaB by this cytokine through a mechanism that most probably involves the phosphorylation of IkappaB. The inability of these PKC isotypes to directly phosphorylate IkappaB led to the hypothesis that zetaPKC may use a putative IkappaB kinase to functionally inactivate IkappaB. Recently several groups have molecularly characterized and cloned two IkappaB kinases (IKKalpha and IKKbeta) that phosphorylate the residues in the IkappaB molecule that serve to target it for ubiquitination and degradation. In this study the possibility that different PKCs may control NF-kappaB through the activation of the IKKs is addressed. AlphaPKC as well as the atypical PKCs bind to the IKKs in vitro and in vivo. In addition, overexpression of zetaPKC positively modulates IKKbeta activity but not that of IKKalpha. In contrast, the transfection of a zetaPKC dominant negative mutant severely impairs the activation of IKKbeta but not IKKalpha in TNF-alpha-stimulated cells. Cell stimulation with phorbol 12-myristate 13-acetate activates IKKbeta, which is entirely dependent on the activity of alphaPKC but not that of the atypical isoforms. In contrast, the inhibition of alphaPKC does not affect the activation of IKKbeta by TNF-alpha. Interestingly, recombinant active zetaPKC and alphaPKC are able to stimulate in vitro the activity of IKKbeta but not that of IKKalpha. In addition, evidence is presented that recombinant zetaPKC directly phosphorylates IKKbeta in vitro, involving Ser177 and Ser181. Collectively, these results demonstrate a critical role for the PKC isoforms in the NF-kappaB pathway at the level of IKKbeta activation and IkappaB degradation (Lallena, 1999).

IkappaB kinase (IKK) phosphorylates IkappaB inhibitory proteins, causing their degradation and activation of transcription factor NF-kappaB, a master activator of inflammatory responses. IKK is composed of three subunits: IKKalpha and IKKbeta, which are highly similar protein kinases, and IKKgamma, a regulatory subunit. In mammalian cells, phosphorylation of two sites at the activation loop of IKKbeta is essential for activation of IKK by tumor necrosis factor and interleukin-1. Elimination of equivalent sites in IKKalpha, however, does not interfere with IKK activation. Thus, IKKbeta, not IKKalpha, is the target for proinflammatory stimuli. Once activated, IKKbeta autophosphorylates at a carboxyl-terminal serine cluster. Such phosphorylation decreases IKK activity and may prevent prolonged activation of the inflammatory response (Delhase, 1999).

The two members of the atypical protein kinase C (aPKC) subfamily of isozymes (zetaPKC and lambda/iotaPKC) are involved in the control of NF-kappaB through IKKbeta activation. The previously described aPKC-binding protein, p62, selectively interacts with RIP but not with TRAF2 in vitro and in vivo. p62 bridges the aPKCs to RIP, whereas the aPKCs link IKKbeta to p62. In this way, a signaling cascade of interactions is established from the TNF-R1 involving TRADD/RIP/p62/aPKCs/IKKbeta. These observations define a novel pathway for the activation of NF-kappaB involving the aPKCs and p62. Consistent with this model, the expression of a dominant-negative mutant lambda/iotaPKC impairs RIP-stimulated NF-kappaB activation. In addition, the expression of either an N-terminal aPKC-binding domain of p62, or its C-terminal RIP-binding region are sufficient to block NF-kappaB activation. Furthermore, transfection of an antisense construct of p62 severely abrogates NF-kappaB activation. Together, these results demonstrate that the interaction of p62 with RIP serves to link the atypical PKCs to the activation of NF-kappaB by the TNFalpha signaling pathway (Sanz, 1999).

A novel PMA-inducible IkappaB kinase complex has been identified, distinct from the well-characterized high-molecular weight IkappaB kinase complex containing IKKalpha, IKKbeta, and IKKgamma. Described here is one kinase from this complex, which has been designated IKKepsilon. Although recombinant IKKepsilon directly phosphorylates only serine 36 of IkappaBalpha, the PMA-activated endogenous IKKepsilon complex phosphorylates both critical serine residues. Remarkably, this activity is due to the presence of a distinct kinase in this complex. A dominant-negative mutant of IKKepsilon blocks induction of NF-kappaB by both PMA and activation of the T cell receptor but has no effect on the activation of NF-kappaB by TNFalpha or IL-1. These observations indicate that the activation of NF-kappaB requires multiple distinct IkappaB kinase complexes, which respond to both overlapping and discrete signaling pathways (Peters, 2000).

To identify potential homologs of IKKalpha and IKKbeta, BLAST searches of GenBank and various EST databases were performed. The cDNA KIAA0151 shares significant homology with IKKalpha and IKKbeta, extending from the amino terminus of the protein to 175 amino acid residues past the kinase domain. This EST maps to human chromosome 1, and corresponding ESTs have been isolated from brain, germ cell, heart, kidney, tonsil, and breast cDNA libraries. A Northern blot analysis confirms that the 3.2 kb KIAA0151 transcript is found in a number of tissues, including those described above, but is particularly highly expressed in the thymus, spleen, and peripheral blood leukocytes. Alignment of the amino acid sequence encoded by KIAA0151 with that of IKKalpha and IKKbeta reveals 33% and 31% amino acid identity with IKKalpha and IKKbeta, respectively, within the kinase domain, and 27% amino acid identity with each throughout the entire sequence. The KIAA0151 gene product has a predicted molecular mass of 80 kDa but does not appear to have the canonical leucine zipper motif found in IKKalpha and IKKbeta. However, analysis of the KIAA0151 amino acid sequence with the Paircoil program has revealed the existence of a coiled coil multimerization domain. The existence of such a multimerization domain is further supported by the ability of the transfected KIAA0151 gene product to interact with itself. Similar to IKKalpha and IKKbeta, the KIAA0151 gene product also contains a possible helix-loop-helix (HLH) region in its C terminus. Based on the sequence similarity between KIAA0151 and IKKalpha and IKKbeta, and the functional evidence provided below, the KIAA0151 gene product has been renamed IKKepisilon (Peters, 2000).

TRAF6 is a signal transducer in the NF-kappaB pathway that activates IkappaB kinase (IKK) in response to proinflammatory cytokines. A heterodimeric protein complex that links TRAF6 to IKK activation has been purified. Peptide mass fingerprinting analysis reveals that this complex is composed of the ubiquitin conjugating enzyme Ubc13 and the Ubc-like protein Uev1A. TRAF6, a RING domain protein, functions together with Ubc13/Uev1A to catalyze the synthesis of unique polyubiquitin chains linked through lysine-63 (K63) of ubiquitin. Blockade of this polyubiquitin chain synthesis, but not inhibition of the proteasome, prevents the activation of IKK by TRAF6. These results unveil a new regulatory function for ubiquitin, in which IKK is activated through the assembly of K63-linked polyubiquitin chains (Deng, 2000).

TRAF6 is a signal transducer that activates IkappaB kinase (IKK) and Jun amino-terminal kinase (JNK) in response to pro-inflammatory mediators such as interleukin-1 (IL-1) and lipopolysaccharides (LPS). IKK activation by TRAF6 requires two intermediary factors, TRAF6-regulated IKK activator 1 (TRIKA1) and TRIKA2. TRIKA1 is a dimeric ubiquitin-conjugating enzyme complex composed of Ubc13 and Uev1A (or the functionally equivalent Mms2). This Ubc complex, together with TRAF6, catalyses the formation of a Lys 63 (K63)-linked polyubiquitin chain that mediates IKK activation through a unique proteasome-independent mechanism. TRIKA2 is composed of TAK1, TAB1 and TAB2, a protein kinase complex previously implicated in IKK activation through an unknown mechanism. TAK1 kinase complex phosphorylates and activates IKK in a manner that depends on TRAF6 and Ubc13-Uev1A. Moreover, the activity of TAK1 to phosphorylate MKK6, which activates the JNK-p38 kinase pathway, is directly regulated by K63-linked polyubiquitination. Evidence is provided that TRAF6 is conjugated by the K63 polyubiquitin chains. These results indicate that ubiquitination has an important regulatory role in stress response pathways, including those of IKK and JNK (Wang, 2001).

TAK1 was thought to activate IKK through the intermediary kinase NIK; however, genetic studies have shown that NIK is not involved in IKK activation by most stimuli, including IL-1ß. Thus, it is unlikely that NIK is the link between TAK1 and IKK. Instead, the data strongly suggest that TAK1 is an IKKK that phosphorylates and activates IKK in the TRAF6 pathway. How TAK1 itself is activated has been unknown. Although recombinant TAK1 can be activated by TAB1, endogenous TAK1 is inactive even though it is always associated with TAB1 and TAB2. Thus, association of TAB1 with TAK1 per se cannot account for the activation of the endogenous TAK1 complex. The results indicating that TRAF6 is ubiquitinated, and that the formation of K63-linked polyubiquitin chains directly activates the endogenous TAK1 complex in a biochemically well defined system provide a new mechanism for the activation of TAK1. According to this mechanism, stimulation of cells with IL-1ß leads to the oligomerization of TRAF6, which triggers its ubiquitination through the action of Ubc13-Uev1A. IL-1ß treatment also triggers the release of TAB2 from a membraneous location to cytoplasm, where it binds to ubiquitinated TRAF6 as well as TAK1. Exactly how TAK1 is activated by ubiquitinated TRAF6 is unclear. One possibility is that polyubiquitination may convert the TRAF6-TAB2 complex into an activator of TAK1 (for example, through a conformational change). Alternatively, the K63-linked polyubiquitin chains attached to TRAF6 may reach into TAK1 to activate it as a result of complex formation among TRAF6, TAB2 and TAK1. It is also formally possible that there is a transient but undetectable level of polyubiquitination on TAK1, and that this dynamic ubiquitination of TAK1 leads directly to its activation. Notwithstanding this uncertainty concerning the detailed mechanism of TAK1 activation, these results demonstrate a crucial role of K63-linked polyubiquitination in TAK1 activation (Wang, 2001).

Ubiquitin-dependent activation of TAK1 offers a possible solution to a conceptual problem inherent in kinase cascades: if kinase C is activated by kinase B, which is in turn activated by kinase A, how is kinase A initially activated? By definition, the first kinase cannot be activated by another kinase, so the initial activation has to be a kinase-independent mechanism. The formation of K63-linked polyubiquitin chains may provide such a mechanism, at least in the case of TAK1 activation. Activated TAK1 then phosphorylates IKK and MKK6, leading to the activation of NF-kappaB and JNK-p38 kinase pathways, respectively. These results raise the possibility that polyubiquitination through K63 of ubiquitin may be a general mechanism for the regulation of stress kinase pathways, including those of IKK and JNK (Wang, 2001).

The transcription factor NF-kappaB is essential for survival of many cell types. However, cells can undergo apoptosis despite the concurrent NF-kappaB activation. It is unknown how the protection conveyed by NF-kappaB is overridden during apoptosis. IkappaB kinase (IKK) ß is specifically proteolyzed by Caspase-3-related caspases at aspartic acid residues 78, 242, 373, and 546 during tumor necrosis factor (TNF)-alpha-induced apoptosis. Proteolysis of IKKß eliminates its enzymatic activity, interfers with IKK activation, and promotes TNF-alpha killing. Point mutations that abrogate IKKß proteolysis generate a caspase-resistant IKKß mutant, which suppresses TNF-alpha-induced apoptosis. Thus, this study demonstrates that TNF-alpha-induced apoptosis requires caspase-mediated proteolysis of IKKß (Tang, 2001).

What is the mechanism by which the UC-IKKß mutant suppresses TNF-alpha-induced apoptosis? One of the possibilities is that it functions through prolonged induction of NF-kappaB-controlled antiapoptotic proteins. It is envisioned that apoptotic death of a cell requires amplification of the caspase cascade, an event that may depend on destruction of cellular survival factors. Treatment with TNF-alpha triggers rapid activation and reactivation of IKK and NF-kappaB. This results in production of antiapoptotic proteins, such as c-IAP1, which inhibit caspases. The life balance is maintained and cells survive. The addition of cycloheximide (CHX) reduces the production of antiapoptotic proteins, allowing activation of caspases such as Casp3. Activated Casp3 in turn cleaves IKKß, blunting IKK reactivation and subsequent production of antiapoptotic proteins. Activated Casp3 also cleaves antiapoptotic proteins, including c-IAP1. Therefore, the caspase cascade is amplified, switching the life balance toward death. In contrast, the uncleavable IKKß (IKK UCß) mutant is resistant to caspase-mediated proteolysis and can be reactivated over a prolonged period of time. Since 10 µg/ml CHX reduces, but does not completely block protein synthesis, activation of NF-kappaB by the UC IKKß mutant allows the accumulation of antiapoptotic proteins at a level that is sufficient to inhibit caspases. As a result, amplification of the caspase cascade and cell death is suppressed (Tang, 2001).

The transcription factor NF-kappaB regulates genes involved in innate and adaptive immune response, inflammation, apoptosis, and oncogenesis. Proinflammatory cytokines induce the activation of NF-kappaB in both transient and persistent phases. The mechanism for this biphasic NF-kappaB activation has been investigated. MEKK3 is essential in the regulation of rapid activation of NF-kappaB, whereas MEKK2 is important in controlling the delayed activation of NF-kappaB in response to stimulation with the cytokines TNF-alpha and IL-1alpha. MEKK3 is involved in the formation of the IkappaBalpha:NF-kappaB/IKK complex, whereas MEKK2 participates in assembling the IkappaBbeta:NF-kappaB/IKK complex; these two distinct complexes regulate the proinflammatory cytokine-induced biphasic NF-kappaB activation. Thus, this study reveals a novel mechanism in which different MAP3K and IkappaB isoforms are involved in specific complex formation with IKK and NF-kappaB for regulating the biphasic NF-kappaB activation. These findings provide further insight into the regulation of cytokine-induced specific and temporal gene expression (Schmidt, 2003).

To date, much effort has focused on the identification of MAPK cascades that are activated by MAP3K. However, the upstream regulators of MEKK2/3 have not been identified, and some of them are likely to be activators of MEKK2/3 in regulation of the biphasic NF-κB activation. The data suggest that one of the possible steps by which MEKK2/3 proteins organize the complex formation is the phosphorylation of Ser177 and Ser181 of the activation loop in IKK2. Phosphorylation of Ser177 and Ser181 of the activation loop in IKK2 moves the activation loop away from the catalytic pocket to allow its interaction with ATP and polypeptide substrates (IκB:NF-κB), thus allowing the transient formation of MEKK2:IKK:IκBβ:NF-κB and MEKK3:IKK:IκBα:NF-κB complexes at similar time points after cytokine stimulation. This may explain why the kinase activity of MEKK2/3 is required for the inducible complex formation between MEKK2/3:IKK:IκB:NF-κB whereas the kinase activity of IKK is dispensable. However, the data do not exclude that other, as yet unidentified, proteins are also involved in this complex formation. The mechanisms regulating the specific interactions between IKK:IκBβ:NF-κB and MEKK2 and between IKK:IκBα:NF-κB and MEKK3 are unclear. It has been shown that 14-3-3epsilon and 14-3-3ζ bind to MEKK2/3 as scaffold for protein-protein interactions. Although they do not appear to exert any direct influence on the kinase activity of MEKK3 in vitro, one may speculate that these scaffold proteins might directly or indirectly influence the formation of MEKK2/3:IKK:IκB:NF-κB complexes in regulation of the biphasic NF-κB activation. κB-Ras proteins that are associated only with the NF-κB:IκBβ complex may provide an explanation for the slower phosphorylation and degradation of IκBβ compared with IκBα (Schmidt, 2003).

The transcription factor NF-kappaB is critical for setting the cellular sensitivities to apoptotic stimuli, including DNA damaging anticancer agents. Central to NF-kappaB signaling pathways is NEMO/IKKgamma the regulatory subunit of the cytoplasmic IkappaB kinase (IKK) complex. While NF-kappaB activation by genotoxic stress provides an attractive paradigm for nuclear-to-cytoplasmic signaling pathways, the mechanism by which nuclear DNA damage modulates NEMO to activate cytoplasmic IKK remains unknown. Genotoxic stress is shown to cause nuclear localization of IKK-unbound NEMO via site-specific SUMO-1 attachment. Surprisingly, this sumoylation step is ATM-independent, but nuclear localization allows subsequent ATM-dependent ubiquitylation of NEMO to ultimately activate IKK in the cytoplasm. Thus, genotoxic stress induces two independent signaling pathways: SUMO-1 modification and ATM activation, which work in concert to sequentially cause nuclear targeting and ubiquitylation of free NEMO to permit the NF-kappaB survival pathway. These SUMO and ubiquitin modification pathways may serve as anticancer drug targets (Huang, 2003).

The major heat shock protein, Hsp70, can protect against cell death by directly interfering with mitochondrial apoptosis pathways. However, Hsp70 also sensitizes cells to certain apoptotic stimuli like TNF. Hsp70 promotes TNF killing by specifically binding the coiled-coil domain of IkappaB kinase gamma (IKKgamma) to inhibit IKK activity and consequently inhibit NF-kappaB-dependent antiapoptotic gene induction. An IKKgamma mutant, which interacts with Hsp70, competitively inhibits the Hsp70-IKKgamma interaction and relieves heat-mediated NF-kappaB suppression. Because Hsp70 inhibits NF-kappaB activity without directly binding NF-kappaB, this indicates that Hsp70 impairs NF-kappaB signaling upstream of NF-kappaB (p65). Because activation of NF-kappaB requires phosphorylation of IkappaB, the effects of Hsp70 on the phosphorylation of IkappaB was examined using specific antibodies to examine phosphorylated and unphosphorylated IkappaBalpha proteins. The results showed that Hsp70 inhibits TNF-induced IkappaBalpha phosphorylation. Depletion of Hsp70 expression with RNA interference rescues TNF-mediated cell death. Although TNF may or may not be sufficient to trigger apoptosis on its own, TNF-triggered apoptosis was initiated or made worse when Hsp70 expression increased to high levels to disrupt NF-kappaB signaling. These results provide significant novel insights into the molecular mechanism for the pro-apoptotic behavior of Hsp70 in death-receptor-mediated cell death (Ran, 2004).

Muscle wasting accompanies aging and pathological conditions ranging from cancer, cachexia, and diabetes to denervation and immobilization. Activation of NF-kappaB, through muscle-specific transgenic expression of activated IkappaB kinase ß (MIKK), causes profound muscle wasting that resembles clinical cachexia. In contrast, no overt phenotype was seen upon muscle-specific inhibition of NF-kappaB through expression of IkappaBalpha superrepressor (MISR). Muscle loss was due to accelerated protein breakdown through ubiquitin-dependent proteolysis. Expression of the E3 ligase MuRF1, a mediator of muscle atrophy, is increased in MIKK mice. Pharmacological or genetic inhibition of the IKKß/NF-kappaB/MuRF1 pathway reverses muscle atrophy. Denervation- and tumor-induced muscle loss are substantially reduced and survival rates improved by NF-kappaB inhibition in MISR mice, consistent with a critical role for NF-kappaB in the pathology of muscle wasting and establishing it as an important clinical target for the treatment of muscle atrophy (Cai, 2004).

The discovery that NF-κB activation is sufficient to cause skeletal muscle atrophy in vivo and that blockade of the NF-κB pathway can ameliorate atrophy suggests a new set of drug targets for clinical intervention during cachexia, cancer, AIDS, and other settings of atrophy. For example, the inhibition of atrophy with high doses of sodium salicylate suggests that more specific inhibitors of IKKβ might be useful to block atrophy -- sodium salicylate itself has a range of other targets, and the high doses of it required for IKKβ inhibition are not well tolerated due to side effects. MuRF1 has already been suggested as a novel clinical target, and the data in this study help to put the previous findings in a larger signaling context. Further study of the IKKβ/NF-κB/MuRF1 pathway will help to uncover other clinically useful targets. This is critically important, because unfortunately, there are currently no drugs approved for the treatment of skeletal muscle atrophy (Cai, 2004).

IKKβ-dependent NF-κB activation plays a key role in innate immunity and inflammation, and inhibition of IKKβ has been considered as a likely anti-inflammatory therapy. Surprisingly, however, mice with a targeted IKKβ deletion in myeloid cells are more susceptible to endotoxin-induced shock than control mice. Increased endotoxin susceptibility is associated with elevated plasma IL-1β as a result of increased pro-IL-1β processing, which was also seen upon bacterial infection. In macrophages enhanced pro-IL-1β processing depends on caspase-1, whose activation is inhibited by NF-κB-dependent gene products. In neutrophils, however, IL-1β secretion is caspase-1 independent and depends on serine proteases, whose activity is also inhibited by NF-κB gene products. Prolonged pharmacologic inhibition of IKKβ also augments IL-1β secretion upon endotoxin challenge. These results unravel an unanticipated role for IKKβ-dependent NF-κB signaling in the negative control of IL-1β production and highlight potential complications of long-term IKKβ inhibition (Greten, 2007).

Table of contents

cactus: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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