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IkappaB stability, phosphorylation and degradation: IkappaB kinases

The alpha catalytic subunit of casein kinase II (CkII) associates with and phosphorylates IkappaBalpha (See Drosophila Casein kinase II ). Deletion mutants of IkappaBalpha localize phosphorylation to the C-terminal PEST domain of IkappaBalpha. Point mutation of residues T-291, S-283, and T-299 dramatically reduces phosphorylation of IkappaBalpha by the kinase in vitro. Cells that stably expresses wild-type IkappaBalpha (wtIkappaB), double-point-mutated IkappaBalpha (T291A, S283A), or triple-point-mutated IkappaBalpha (T291A, S283A, T299A) under the control of the tetracycline-responsive promoter were generated. Constitutive phosphorylation of the triple point mutant is eliminated in vivo. Mutation of the CkII sites in IkappaBalpha result in a protein with increased intrinsic stability. Together with results demonstrating a role for N-terminal sites in inducer-mediated phosphorylation and degradation of IkappaBalpha, these studies indicate that CkII sites in the C-terminal PEST domain are important for constitutive phosphorylation and intrinsic stability of IkappaBalpha (Lin, 1996).

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) by inflammatory cytokines requires the successive action of NF-kappaB-inducing kinase (NIK) and IkappaB kinase-alpha (IKK-alpha). A widely expressed protein kinase has been identified that is 52 percent identical to IKK-alpha. IkappaB kinase-beta (IKK-beta) activates NF-kappaB when overexpressed and phosphorylates serine residues 32 and 36 of IkappaB-alpha and serines 19 and 23 of IkappaB-beta. The activity of IKK-beta is stimulated by tumor necrosis factor and interleukin-1 treatment. IKK-alpha and IKK-beta form heterodimers that interact with NIK. Overexpression of a catalytically inactive form of IKK-beta blocks cytokine-induced NF-kappaB activation. Thus, an active IkappaB kinase complex may require three distinct protein kinases (Woronicz, 1997).

Expression of NF-kappaB in the nucleus occurs after signal-induced phosphorylation, ubiquitination, and proteasome-mediated degradation of IkappaBalpha. The induced proteolysis of IkappaBalpha unmasks the nuclear localization signal within NF-kappaB, allowing its rapid migration into the nucleus, where it activates the transcription of many target genes. At present, the identity of the IkappaBalpha kinase(s) that triggers the first step in IkappaBalpha degradation remains unknown. The 90-kDa ribosomal S6 kinase, or pp90(rsk) lies downstream of mitogen-activated protein (MAP) kinase in the well characterized Ras-Raf-MEK-MAP kinase pathway that is induced by various growth factors and phorbol ester. pp90(rsk), but not pp70(S6K) or MAP kinase, phosphorylates the regulatory N terminus of IkappaBalpha principally on serine 32 and triggers effective IkappaBalpha degradation in vitro. When co-expressed in vivo in COS cells, IkappaBalpha and pp90(rsk) readily assemble into a complex that is immunoprecipitated with antibodies specific for either partner. While phorbol ester produces rapid activation of pp90(rsk), in vivo, other potent NF-kappaB inducers, including tumor necrosis factor alpha and the Tax transactivator of human T-cell lymphotrophic virus, type I, fail to activate pp90(rsk). These data suggest that more than a single IkappaBalpha kinase exists within the cell and that these IkappaBalpha kinases are differentially activated by different NF-kappaB inducers (Ghoda, 1997).

Regulation of the IkappaB alpha and IkappaB beta proteins is critical for modulating NF-kappaB-directed gene expression. Both IkappaB alpha and IkappaB beta are substrates for cellular kinases that phosphorylate the amino and carboxy termini of these proteins and regulate their function. In this study, a biochemical fractionation scheme was utilized to purify a kinase activity that phosphorylates residues in the amino and carboxy termini of both IkappaB alpha and IkappaB beta. Peptide microsequence analysis by capillary high-performance liquid chromatography ion trap mass spectroscopy reveals that this kinase is the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PK phosphorylates serine residue 36 but not serine residue 32 in the amino terminus of IkappaB alpha and also phosphorylates threonine residue 273 in the carboxy terminus of this protein. To determine the biological relevance of DNA-PK phosphorylation of IkappaB alpha, murine severe combined immunodeficiency (SCID) cell lines that lack the DNA-PKcs gene were analyzed. Gel retardation analysis using extract prepared from these cells demonstrates constitutive nuclear NF-kappaB DNA binding activity, which is not detected in extracts prepared from SCID cells complemented with the human DNA-PKcs gene. Furthermore, IkappaB alpha that is phosphorylated by DNA-PK is a more potent inhibitor of NF-kappaB binding than nonphosphorylated IkappaB alpha. These results suggest that DNA-PK phosphorylation of IkappaB alpha increases its interaction with NF-kappaB to reduce NF-kappaB DNA binding properties (Liu, 1998).

There are three known consequences of Tumor necrosis factor receptor (TNFR) activation: (1) inductior of apoptosis through TRADD and FADD (see Caspase), (2) activation of JNK (see Basket), and (3) activation of transcription factor NF-kappaB. Activation of NF-kappaB by tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the NF-kappaB-inducing kinase (NIK). In a yeast two-hybrid screen for NIK-interacting proteins, a protein kinase has been identified previously known as CHUK. Overexpression of CHUK activates a NF-kappaB-dependent reporter gene. A catalytically inactive mutant of CHUK is a dominant-negative inhibitor of TNF-, IL-1-, TRAF- (the TNF receptor-associated factor), and NIK-induced NF-kappaB activation. CHUK associates with the NF-kappaB inhibitory protein, IkappaB-alpha, in mammalian cells. CHUK specifically phosphorylates IkappaB-alpha on both serine 32 and serine 36, modifications that are required for targeted degradation of IkappaB-alpha via the ubiquitin-proteasome pathway. This phosphorylation of IkappaB-alpha is greatly enhanced by NIK costimulation. Thus, CHUK is a NIK-activated IkappaB-alpha kinase that links TNF- and IL-1-induced kinase cascades to NF-kappaB activation (Regnier, 1997).

IKKalpha; also called IKK1, the gene encoding inhibitor of kappa B (IkappaB) kinase alpha was disrupted by gene targeting. IKKalpha-deficient mice die perinatally. In IKKalpha-deficient fetuses, limb outgrowth is severely impaired despite unaffected skeletal development. The epidermal cells in IKKalpha-deficient fetuses are highly proliferative with dysregulated epidermal differentiation. In the basal layer, degradation of IkappaB and nuclear localization of nuclear factor kappa B (NF-kappaB) are not observed. Thus, IKKalpha is essential for NF-kappaB activation in the limb and skin during embryogenesis. In contrast, there is no impairment of NF-kappaB activation induced by either interleukin-1 or tumor necrosis factor-alpha in IKKalpha-deficient embryonic fibroblasts and thymocytes, indicating that IKKalpha is not essential for cytokine-induced activation of NF-kappaB (Takeda, 1999).

The oligomeric IkappaB kinase (IKK) is composed of three polypeptides: IKKalpha and IKKbeta, the catalytic subunits, and IKKgamma, a regulatory subunit. IKKalpha and IKKbeta are similar in structure and thought to have similar function-phosphorylation of the IkappaB inhibitors in response to proinflammatory stimuli. Such phosphorylation leads to degradation of IkappaB and activation of nuclear factor kappaB transcription factors. The physiological function of these protein kinases was explored by analysis of IKKalpha-deficient mice. IKKalpha is not required for activation of IKK and degradation of IkappaB by proinflammatory stimuli. Instead, loss of IKKalpha interfers with multiple morphogenetic events, including limb and skeletal patterning and proliferation and differentiation of epidermal keratinocytes (Hu, 1999).

Phosphorylation of inhibitor of kappa B (IkappaB) proteins is an important step in the activation of the transcription nuclear factor kappa B (NF-kappaB) and requires two IkappaB kinases, IKK1 (IKKalpha) and IKK2 (IKKbeta). Mice that are devoid of the IKK2 gene have extensive liver damage from apoptosis and die as embryos, but these mice can be rescued by the inactivation of the gene encoding tumor necrosis factor receptor 1. Mouse embryonic fibroblast cells, isolated from IKK2(-/-) embryos, show a marked reduction in tumor necrosis factor-alpha (TNF-alpha)- and interleukin-1alpha-induced NF-kappaB activity and an enhanced apoptosis in response to TNF-alpha. IKK1 associates with NF-kappaB essential modulator (IKKgamma/IKKAP1), another component of the IKK complex. These results show that IKK2 is essential for mouse development and cannot be substituted with IKK1 (Li, 1999a).

IkappaB kinases 1 and 2 (IKKs: IKK1 and IKK2) are two putative IkappaBalpha kinases involved in NF-kappaB activation. To examine the in vivo functions of IKK1, IKK1-deficient mice were generated. The mutant mice are perinatally lethal and exhibit a wide range of developmental defects. Newborn mutant mice have shiny, taut, and sticky skin without whiskers. Histological analysis shows thicker epidermis, which is unable to differentiate. Limbs and tail are wrapped inside the skin and do not extend properly out of the body trunk. Skeleton staining reveals a cleft secondary palate, split sternebra 6, and deformed incisors. NF-kappaB activation mediated by TNFalpha and IL-1 is diminished in IKK1-deficient mouse embryonic fibroblast (MEF) cells. The IKK complex in the absence of IKK1 is capable of phosphorylating IkappaBalpha and IkappaBbeta in vitro. These results support a role for IKK1 in NF-kappaB activation and uncover its involvement in skin and skeleton development. It is concluded further that the two related kinases IKK1 and IKK2 have distinct functions and can not be substituted for each other's functions (Li, 1999b).

NF-κB is activated in response to proinflammatory stimuli, infections, and physical stress. While activation of NF-κB by many stimuli depends on the IκB kinase (IKK) complex, which phosphorylates IκBs at N-terminal sites, the mechanism of NF-κB activation by ultraviolet (UV) radiation remained enigmatic, since it is IKK independent. UV-induced NF-κB activation has been shown to depend on phosphorylation of IκBα at a cluster of C-terminal sites that are recognized by CK2 (formerly casein kinase II). Furthermore, CK2 activity toward IκB is UV inducible through a mechanism that depends on activation of p38 MAP kinase. Inhibition of this pathway prevents UV-induced IκBα degradation and increases UV-induced cell death. Thus, the p38-CK2-NF-κB axis is an important component of the mammalian UV response (Kato, 2003).

IkappaB kinases are targeted by NF-kappaB-inducing kinase downstream of TNF and TRAFs

TNF-induced activation of the transcription factor NF-kappaB and the c-jun N-terminal kinase (JNK/SAPK) requires TNF receptor-associated factor 2 (TRAF2). The NF-kappaB-inducing kinase (NIK, a different protein than Nck interacting kinase, a SH2-SH3 oncogene) associates with TRAF2 and mediates TNF activation of NF-kappaB. NIK interacts with additional members of the TRAF family and this interaction requires the conserved WKI motif within the TRAF domain. The role of NIK in JNK activation by TNF was investigated. Whereas overexpression of NIK potently induces NF-kappaB activation, it fails to stimulate JNK activation. A kinase-inactive mutant of NIK is a dominant negative inhibitor of NF-kappaB activation but does not suppress TNF- or TRAF2-induced JNK activation. Thus, TRAF2 is the bifurcation point of two kinase cascades leading to activation of NF-kappaB and JNK, respectively (Song, 1997).

Interleukin-1 (IL-1) is a proinflammatory cytokine that has several effects in the inflammation process. When it binds to its cell-surface receptor, IL-1 initiates a signaling cascade that leads to activation of the transcription factor NF-kappaB and is relayed through the protein TRAF6 and a succession of kinase enzymes, including NF-kappaB-inducing kinase (NIK) and I kappaB kinases (IKKs). However, the molecular mechanism by which NIK is activated is not understood. The MAPKK kinase TAK1 (Drosophila homolog: TGF-ß activated kinase 1) acts upstream of NIK in the IL-1-activated signaling pathway and TAK1 associates with TRAF6 during IL-1 signaling. Stimulation of TAK1 causes activation of NF-kappaB, which is blocked by dominant-negative mutants of NIK, and an inactive TAK1 mutant prevents activation of NF-kappaB that is mediated by IL-1 but not by NIK. Activated TAK1 phosphorylates NIK, which stimulates IKK-alpha activity. These results indicate that TAK1 links TRAF6 to the NIK-IKK cascade in the IL-1 signaling pathway (Ninomiya-Tsuji, 1998).

The human homolog of Drosophila Toll (hToll) is a recently cloned receptor of the interleukin 1 receptor (IL-1R) superfamily, and has been implicated in the activation of adaptive immunity. Signaling by hToll occurs through sequential recruitment of the adapter molecule MyD88 (see Drosophila Myd88) and the IL-1R-associated kinase. Tumor necrosis factor receptor-activated factor 6 (TRAF6) and the nuclear factor kappaB (NF-kappaB)-inducing kinase (NIK) are both involved in subsequent steps of NF-kappaB activation. Conversely, a dominant negative version of TRAF6 fails to block hToll-induced activation of stress-activated protein kinase/c-Jun NH2-terminal kinases, thus suggesting an early divergence of the two pathways (Muzio, 1998).

CD27 is a member of the tumor necrosis factor (TNF) receptor superfamily and is expressed on T, B, and NK cells. The signal via CD27 plays pivotal roles in T-T and T-B cell interactions. Overexpression of CD27 activates NF-kappaB and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). Deletion analysis of the cytoplasmic domain of CD27 reveals that the C-terminal PIQEDYR motif is indispensable for both NF-kappaB and SAPK/JNK activation and is also required for the interaction with TNF receptor-associated factor (TRAF) 2 and TRAF5, both of which have been implicated in NF-kappaB activation by members of the TNF-R superfamily. Co-transfection of a dominant negative TRAF2 or TRAF5 blocks NF-kappaB and SAPK/JNK activation induced by CD27. Recently, a TRAF2-interacting kinase has been identified, termed NF-kappaB-inducing kinase (NIK). A kinase-inactive mutant NIK blocks CD27-, TRAF2-, and TRAF5-mediated NF-kappaB and SAPK/JNK activation. These results indicate that TRAF2 and TRAF5 are involved in NF-kappaB and SAPK/JNK activation by CD27, and NIK is a common downstream kinase of TRAF2 and TRAF5 for NF-kappaB and SAPK/JNK activation (Akiba, 1998).

Various members of the tumor necrosis factor (TNF) receptor superfamily activate nuclear factor kappaB (NF-kappaB) and the c-Jun N-terminal kinase (JNK) pathways through their interaction with TNF receptor-associated factors (TRAFs) and NF-kappaB-inducing kinase (NIK). The cytoplasmic domain of receptor activator of NF-kappaB (RANK) interacts with TRAF2, TRAF5, and TRAF6 and its overexpression activates NF-kappaB and JNK pathways. Through a detailed mutational analysis of the cytoplasmic domain of RANK, it has been demonstrated that TRAF2 and TRAF5 bind to consensus TRAF binding motifs located in the C terminus at positions 565-568 and 606-611, respectively. In contrast, TRAF6 interacts with a novel motif located between residues 340 and 358 of RANK. Furthermore, transfection experiments with RANK and its deletion mutants in human embryonic 293 cells reveal that the TRAF6-binding region (340-358), but not the TRAF2 or TRAF5-binding region, is necessary and sufficient for RANK-induced NF-kappaB activation. Moreover, a kinase mutant of NIK (NIK-KM) inhibits RANK-induced NF-kappaB activation. However, RANK-mediated JNK activation required a distal portion (427-603) of RANK containing the TRAF2-binding domain. Thus, these results indicate that RANK interacts with various TRAFs through distinct motifs and activates NF-kappaB via a novel TRAF6 interaction motif, which then activates NIK, thus leading to NF-kappaB activation, whereas RANK most likely activates JNK through a TRAF2-interacting region in RANK (Darnay, 1999).

Interleukin-1 (IL-1) is a proinflammatory cytokine that produces several effects in the inflammation process. When it binds to its cell-surface receptor, IL-1 initiates a signaling cascade that leads to activation of the transcription factor NF-kappaB and is relayed through the protein TRAF6 and a succession of kinase enzymes, including NF-kappaB-inducing kinase (NIK) and I kappaB kinases (IKKs). However, the molecular mechanism by which NIK is activated is not understood. The MAPKK kinase TAK1 acts upstream of NIK in the IL-1-activated signaling pathway and TAK1 associates with TRAF6 during IL-1 signaling. Stimulation of TAK1 causes activation of NF-kappaB, which is blocked by dominant-negative mutants of NIK, and an inactive TAK1 mutant prevents activation of NF-kappaB that is mediated by IL-1 but not by NIK. Activated TAK1 phosphorylates NIK, which stimulates IKK-alpha activity. These results indicate that TAK1 links TRAF6 to the NIK-IKK cascade in the IL-1 signaling pathway (Ninomiya-Tsuji, 1999).

The activation of NF-kappaB by receptors in the tumor necrosis factor (TNF) receptor and Toll/interleukin-1 (IL-1) receptor families requires the TRAF family of adaptor proteins. Receptor oligomerization causes the recruitment of TRAFs to the receptor complex, followed by the activation of a kinase cascade that results in the phosphorylation of IkappaB. TANK is a TRAF-binding protein that can inhibit the binding of TRAFs to receptor tails and can also inhibit NF-kappaB activation by these receptors. However, TANK also displays the ability to stimulate TRAF-mediated NF-kappaB activation. The mechanism by which TANK modulates TRAF2 function has not been well defined. For example, it was not known whether the synergy observed between TANK and TRAF2 reflect action by each protein on the same downstream cascade of molecules, namely NIK and the IKK complex, or whether TANK affects an undefined parallel pathway that also leads to IkappaB phosphorylation. Furthermore, it was unknown whether TANK could serve as a cofactor for other TRAF family members that also activate NFkappa-B, such as TRAF5 and TRAF6. While TANK modulates TRAF2-mediated NF-kappaB activation, the question of whether TANK affects JNK1 activation by TRAF2 has not been addressed. Because of the lack of homology of TANK to any known protein, its mechanism of action is not predictable. The mechanism of the stimulatory activity of TANK has been investigated. TANK interacts with TBK1 (TANK-binding kinase 1), a novel IKK-related kinase that can activate NF-kappaB in a kinase-dependent manner. TBK1, TANK and TRAF2 can form a ternary complex, and complex formation appears to be required for TBK1 activity. Kinase-inactive TBK1 inhibits TANK-mediated NF-kappaB activation but does not block the activation mediated by TNF-alpha, IL-1 or CD40. The TBK1-TANK-TRAF2 signaling complex functions upstream of NIK and the IKK complex and represents an alternative to the receptor signaling complex for TRAF-mediated activation of NF-kappaB (Pomerantz, 1999).

One receptor-ligand pair that could mediate NF-kappaB activation during mammary gland development consists of RANK (receptor activator of NFkappa-B), a member of the TNF receptor (TNFR) family and RANK ligand (RANKL, also known as OPGL, ODF, and TRANCE), a member of the TNF family. As its namesake indicates, RANK is an efficient NF-kappaB activator. Although RANK and RANKL were originally characterized as playing important roles in lymphocyte and osteoclast differentiation and activation, they were recently found to be essential for mammary gland development. The biochemical pathway by which RANK controls mammary gland development has not been defined. IKKalpha activity is required for NF-kappaB activation. To identify functions of the IKKalpha subunit of IkappaB kinase that require catalytic activity, an IkkalphaAA knockin allele was created containing alanines instead of serines in the activation loop. IkkalphaAA/AA mice are healthy and fertile, but females display a severe lactation defect due to impaired proliferation of mammary epithelial cells. IKKalpha activity is required for NF-kappaB activation in mammary epithelial cells during pregnancy and in response to RANK ligand but not TNFalpha. IKKalpha and NF-kappaB activation are also required for optimal cyclin D1 induction. Defective RANK signaling or cyclin D1 expression results in the same phenotypic effect as the IkkalphaAA mutation, which is completely suppressed by a mammary specific cyclin D1 transgene. Thus, IKKalpha is a critical intermediate in a pathway that controls mammary epithelial proliferation in response to RANK signaling via cyclin D1 (Cao, 2001).

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

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