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IkappaB stability, phosphorylation and degradation: General

Activation of the transcription factor NF-kappaB is a paradigm for signal transduction through the ubiquitin-proteasome pathway: ubiquitin-dependent degradation of the transcriptional inhibitor IkappaB in response to cell stimulation. A major issue in this context is the nature of the recognition signal and the targeting enzyme involved in the proteolytic process. Following a stimulus-dependent phosphorylation, and while associated with NF-kappaB, IkappaB is targeted by a specific ubiquitin-ligase via direct recognition of the signal-dependent phosphorylation site: phosphopeptides corresponding to this site specifically inhibit ubiquitin conjugation of IkappaB and its subsequent degradation. The ligase recognition signal is functionally conserved between IkappaBalpha and IkappaBbeta, and does not involve the nearby ubiquitination site. Microinjection of the inhibitory peptides into stimulated cells abolish NF-kappaB activation in response to TNFalpha and the consequent expression of E-selectin, an NF-kappaB-dependent cell-adhesion molecule. Inhibition of NF-kappaB function by specific blocking of ubiquitin ligase activity provides a novel approach for intervening in cellular processes via regulation of unique proteolytic events (Yaron, 1997).

NF-kappaB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules: IkappaBs. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin-proteasome system to eliminate inhibitory regulators. The pIkappaBalpha-ubiquitin ligase (pIkappaBalpha-E3) has been isolated. This ligase attaches ubiquitin, a small protein that marks other proteins for degradation by the proteasome system, to the phosphorylated NF-kappaB inhibitor pIkappaBalpha. Taking advantage of its high affinity to pIkappaBalpha, this ligase has been isolated from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, the specific component of the ligase that recognizes the pIkappaBalpha degradation motif has been identified as an F-box/WD-domain protein belonging to a recently distinguished family of beta-TrCP/ Slimb (see Drosophila supernumerary limbs) proteins. This component, which is denoted E3RSIkappaB (pIkappaBalpha-E3 receptor subunit), binds specifically to pIkappaBalpha and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RS(IkappaB), which tightly binds pIkappaBalpha but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIkappaBalpha and consequently NF-kappaB activation. E3RS(IkappaB) represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation (Yaron, 1998).

Ubiquitin-mediated proteolysis has a central role in controlling the intracellular levels of several important regulatory molecules, such as cyclins, CKIs, p53, and IkappaBalpha. Many diverse proinflammatory signals lead to the specific phosphorylation and subsequent ubiquitin-mediated destruction of the NF-kappaB inhibitor protein IkappaBalpha. Substrate specificity in ubiquitination reactions is, in large part, mediated by the specific association of the E3-ubiquitin ligases with their substrates. One class of E3 ligases is defined by the recently described SCF complexes, the archetype of which was first described in budding yeast and contains Skp1, Cdc53, and the F-box protein Cdc4. These complexes recognize their substrates through modular F-box proteins in a phosphorylation-dependent manner. A biochemical dissection is described of a novel mammalian SCF complex, SCFbeta-TRCP, that specifically recognizes a 19-amino-acid destruction motif in IkappaBalpha (residues 21-41) in a phosphorylation-dependent manner. This SCF complex also recognizes a conserved destruction motif in beta-catenin, a protein with levels also regulated by phosphorylation-dependent ubiquitination. Endogenous IkappaBalpha-ubiquitin ligase activity cofractionates with SCFbeta-TRCP. Furthermore, recombinant SCFbeta-TRCP assembled in mammalian cells contains phospho-IkappaBalpha-specific ubiquitin ligase activity. These results suggest that an SCFbeta-TRCP complex functions in multiple transcriptional programs by activating the NF-kappaB pathway and inhibiting the beta-catenin pathway (Winston, 1999).

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is controlled by proteolysis of its inhibitory subunit (IkappaB) via the ubiquitin-proteasome pathway. Signal-induced phosphorylation of IkappaBalpha by a large multisubunit complex containing IkappaB kinases is a prerequisite for ubiquitination. Here, FWD1 (a mouse homolog of Slimb/betaTrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with IkappaBalpha only when IkappaBalpha is phosphorylated. The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of IkappaBalpha in concert with IkappaB kinases, resulting in nuclear translocation of NF-kappaB. In addition, FWD1 strikingly evokes the ubiquitination of IkappaBalpha in the in vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of IkappaBalpha. These results suggest that (1) the substrate-specific degradation of IkappaBalpha is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex, and (2) that FWD1 serves as an intracellular receptor for phosphorylated IkappaBalpha. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-kappaB through control of IkappaB protein stability (Hatakeyama, 1999).

The SCF complex containing Skp1, Cul1, and the F-box protein FWD1 (the mouse homologue of Drosophila Slimb and Xenopus beta-TrCP) functions as the ubiquitin ligase for IkappaBalpha. FWD1 associates with Skp1 through the F-box domain and also recognizes the conserved DSGXXS motif of IkappaBalpha. The structural requirements for the interactions of FWD1 with IkappaBalpha and with Skp1 have now been investigated further. The D31A mutation (but not the G33A mutation) in the DSGXXS motif of IkappaBalpha abolishes the binding of IkappaBalpha to FWD1 and its subsequent ubiquitination without affecting the phosphorylation of IkappaBalpha. The IkappaBalpha mutant D31E still exhibits binding to FWD1 and undergoes ubiquitination. These results suggest that, in addition to site-specific phosphorylation at Ser(32) and Ser(36), an acidic amino acid at position 31 is required for FWD1-mediated ubiquitination of IkappaBalpha. Deletion analysis of Skp1 reveals that residues 61-143 of this protein are required for binding to FWD1. In contrast, the highly conserved residues Pro(149), Ile(160), and Leu(164) in the F-box domain of FWD1 are dispensable for binding to Skp1. Together, these data delineate the structural requirements for the interactions among IkappaBalpha, FWD1, and Skp1 that underlie substrate recognition by the SCF ubiquitin ligase complex (Hattori, 1999).

The serine/threonine kinase Akt (also known as protein kinase B, PKB: Drosophila homolog Akt1) is activated by numerous growth-factor and immune receptors through lipid products of phosphatidylinositol (PI) 3-kinase. Akt can couple to pathways that regulate glucose metabolism or cell survival. Akt can also regulate several transcription factors, including E2F, CREB, and the Forkhead family member Daf-16. Akt regulates signaling pathways that lead to induction of the NF-kappaB family of transcription factors in the Jurkat T-cell line. This induction occurs, at least in part, at the level of degradation of the NF-kappaB inhibitor IkappaB, and is specific for NF-kappaB, since other inducible transcription factors are not affected by Akt overexpression. Furthermore, the effect requires the kinase activity and pleckstrin homology (PH) domain of Akt. Also, Akt does not act alone to induce cytokine promoters and NF-kappaB reporters, because signals from other pathways are required to observe the effect. These studies uncover a previously unappreciated connection between Akt and NF-kappaB induction that could have implications for the control of T-cell growth and survival (Kane, 1999).

Small guanosine triphosphatases, typified by the mammalian Ras proteins, play major roles in the regulation of numerous cellular pathways. A subclass of evolutionarily conserved Ras-like proteins has been identified, including a Drosophila homolog, members of which differ from other Ras proteins in containing amino acids at positions 12 and 61 that are similar to those present in the oncogenic forms of Ras. These proteins, kappaB-Ras1 and kappaB-Ras2, interact with the PEST domains of IkappaBalpha and IkappaBbeta [inhibitors of the transcription factor nuclear factor kappa B (NF-kappaB)] and decrease their rate of degradation. In cells, kappaB-Ras proteins are associated only with NF-kappaB:IkappaBbeta complexes and therefore may provide an explanation for the slower rate of degradation of IkappaBbeta compared with IkappaBalpha. The kappaB-Ras proteins specifically associate with PEST domains and influence their degradation. It is therefore possible that they might also regulate the turnover of other PEST domain-containing proteins in addition to IkappaBbeta. In fact, kappaB-Ras proteins are detected in high-molecular weight complexes, distinct from the cytoplasmic NF-kappaB/IkappaB complexes. Identification of these other kappaB-Ras-associated proteins will therefore be crucial in understanding the overall biological role of kappaB-Ras proteins (Fenwick, 2000).

In lysosomes isolated from rat liver and spleen, a percentage of the intracellular inhibitor of the nuclear factor kappa B (IkappaB) can be detected in the lysosomal matrix where it is rapidly degraded. Levels of IkappaB are significantly higher in a lysosomal subpopulation that is active in the direct uptake of specific cytosolic proteins. IkappaB is directly transported into isolated lysosomes in a process that requires binding of IkappaB to the heat shock protein of 73 kDa (hsc73), the cytosolic molecular chaperone involved in this pathway, and to the lysosomal glycoprotein of 96 kDa (lgp96), the receptor protein in the lysosomal membrane. Other substrates for this degradation pathway competitively inhibit IkappaB uptake by lysosomes. Ubiquitination and phosphorylation of IkappaB are not required for the targeting of IkappaB to lysosomes. The lysosomal degradation of IkappaB is activated under conditions of nutrient deprivation. Thus, the half-life of a long-lived pool of IkappaB is 4.4 d in serum-supplemented Chinese hamster ovary cells but only 0.9 d in serum-deprived Chinese hamster ovary cells. This increase in IkappaB degradation can be completely blocked by lysosomal inhibitors. The degradation of IkappaB is increased in Chinese hamster ovary cells exhibiting an increased activity in the hsc73-mediated lysosomal degradation pathway due to overexpression of lamp2 (the human form of lgp96). There are both short- and long-lived pools of IkappaB, and it is the long-lived pool that is subjected to the selective lysosomal degradation pathway. In the presence of antioxidants, the half-life of the long-lived pool of IkappaB is significantly increased. Thus, the production of intracellular reactive oxygen species during serum starvation may be one of the mechanisms mediating IkappaB degradation in lysosomes. This selective pathway for the lysosomal degradation of IkappaB is physiologically important since prolonged serum deprivation results in an increase in the nuclear activity of nuclear factor kappa B. In addition, the response of nuclear factor kappa B to several stimuli increases when this lysosomal pathway of proteolysis is activated (Cuervo, 1998).

Hypoxia, reoxygenation, and the tyrosine phosphatase inhibitor pervanadate activate the transcription factor NF-kappaB; this involves the phosphorylation of the NF-kappaB inhibitor, IkappaB-alpha, on tyrosine 42. This modification does not lead to degradation of IkappaB by the proteasome/ubiquitin pathway, as is seen when cells are stimulated with proinflammatory cytokines. It is currently unknown how tyrosine-phosphorylated IkappaB is removed from NF-kappaB. p85alpha, the regulatory subunit of PI3-kinase, specifically associates through its Src homology 2 domains with tyrosine-phosphorylated IkappaB-alpha in vitro and in vivo after stimulation of T cells with pervanadate. This association could provide a mechanism by which newly tyrosine-phosphorylated IkappaB is sequestered from NF-kappaB. Another mechanism by which PI3-kinase contributes to NF-kappaB activation in response to pervanadate appears to involve its catalytic p110 subunit. This is evident from the inhibition of pervanadate-induced NF-kappaB activation and reporter gene induction by treatment of cells with nanomolar amounts of the PI3-kinase inhibitor wortmannin. The compound has virtually no effect on tumor necrosis factor- and interleukin-1-induced NF-kappaB activities. Wortmannin does not inhibit tyrosine phosphorylation of IkappaB-alpha, nor does it alter the stability of the PI3-kinase complex, but wortmannin does inhibit Akt kinase activation in response to pervanadate. These data suggest that both the regulatory and the catalytic subunit of PI3-kinase play a role in NF-kappaB activation by the tyrosine phosphorylation-dependent pathway (Beraud, 1999).

Arachidonic acid (AA), through its myriad metabolites, is involved in inflammation in a number of ways. AA is produced and released by several cell types, including endothelial cells (EC), and acts on a variety of cells. EC activation plays a key role in inflammation, presumably by modulating the immune response through up- or down-regulation of several genes. AA and its nonmetabolizable analogue, 5,8,11,14-eicosatetraynoic acid (ETYA), inhibit up-regulation of proinflammatory genes in EC. A mechanism has been identified to explain the inhibitory effects: AA and ETYA both inhibit the translocation of nuclear factor-kappaB (NF-kappaB) to the nucleus by blocking the degradation of the inhibitor of NF-kappaB (IkappaB) and thus stabilizing the IkappaB/NF-kappaB complex. To investigate the mechanism whereby AA inhibits up-regulation of genes encoding proinflammatory mediators, an examination was made of the ability of ETYA to inhibit tumor necrosis factor-alpha (TNF-alpha) mediated phosphorylation and degradation of IkappaBalpha. Preincubation of EC with ETYA for 40 min prior to stimulation with TNF-alpha inhibits the phosphorylation and degradation of IkappaBalpha. These findings establish a mechanism by which AA inhibits nuclear translocation of NF-kappaB, thereby explaining its modulatory role in the induction of proinflammatory genes (Stuhlmeier, 1997).

Transformation of B-lineage precursors by the Abelson murine leukemia virus appears to arrest development at the pre-B stage. Abelson-transformed pre-B cell lines generally retain transcriptionally inactive, unrearranged immunoglobulin kappa alleles. Nontransformed pre-B cells expanded from mouse bone marrow efficiently transcribe unrearranged kappa alleles. In addition, they contain activated complexes of the NF-kappa B/Rel transcription factor family, in contrast with their Abelson-transformed counterparts. Using conditionally transformed pre-B cell lines, it hs been shown that the v-abl viral transforming protein, a tyrosine kinase (see Drosophila Abl oncogene), blocks germ-line kappa gene transcription and negatively regulates NF-kappa B/Rel activity. An active v-abl kinase specifically inhibits the NF-kappa B/Rel-dependent kappa intron enhancer, which is implicated in promoting both transcription and rearrangement of the kappa locus. v-abl inhibits the activated state of NF-kappa B/Rel complexes in a pre-B cell via a post-translational mechanism that results in increased stability of the inhibitory subunit I kappa B alpha. This analysis suggests a molecular pathway by which v-abl inhibits kappa locus transcription and rearrangement (Klug, 1994).

Binding of plasma Factor VII/VIIa to the tissue factor (TF) receptor initiates the coagulation protease cascades. TF expression by circulating monocytes is associated with thrombotic and inflammatory complications in a variety of diseases. Transcriptional activation of the human TF gene in monocytic cells exposed to bacterial lipopolysaccharide (LPS) is mediated by binding of c-Rel/p65 heterodimers to a kappa B site in the TF promoter. A family of anti-inflammatory agents, known as the salicylates, inhibited LPS induction of TF activity and TF gene transcription in human monocytes and monocytic THP-1 cells. Furthermore, sodium salicylate blockes the LPS-induced proteolytic degradation of I kappa B alpha, which preventes the nuclear translocation of c-Rel/p65 heterodimers. These results indicate that salicylates inhibite LPS induction of TF gene transcription in monocytic cells by preventing nuclear translocation of c-Rel/p65 heterodimers (Oeth, 1995).

In resting T lymphocytes, the transcription factor NF-kappaB is sequestered in the cytoplasm via interactions with members of the I kappa B family of inhibitors, including IkappaBalpha and IkappaBbeta. During normal T-cell activation, IkappaBalpha is rapidly phosphorylated, ubiquitinated, and degraded by the 26S proteasome, thus permitting the release of functional NF-kappaB. In contrast to its transient pattern of nuclear induction during an immune response, NF-kappaB is constitutively activated in cells expressing the Tax transforming protein of human T-cell leukemia virus type I (HTLV-1). Recent studies indicate that HTLV-1 Tax targets IkappaBalpha to the ubiquitin-proteasome pathway. In addition to acting on IkappaBalpha, Tax stimulates the turnover of IkappaBbeta via a related targeting mechanism. Like IkappaBalpha, Tax-mediated breakdown of IkappaBbeta in transfected T lymphocytes can be blocked either by cell-permeable proteasome inhibitors or by mutation Of IkappaBbeta at two serine residues present within its N-terminal region. Despite the dual specificity of HTLV-1 Tax for IkappaBalpha and IkappaBbeta at the protein level, Tax selectively stimulates NF-kappaB-directed transcription of the IkappaBalpha gene. Consequently, IkappaBbeta protein expression is chronically downregulated in HTLV-1-infected T lymphocytes. These findings with IkappaBbeta provide a potential mechanism for the constitutive activation of NF-kappaB in Tax-expressing cells (McKinsey, 1996).

The HBx protein is a small polypeptide encoded by mammalian hepadnaviruses that is essential for viral infectivity and is thought to play a role in development of hepatocellular carcinoma during chronic hepatitis B virus infection. HBx is a transactivator that stimulates Ras signal transduction pathways in the cytoplasm (See Drosophila Ras) and certain transcription elements in the nucleus. HBx induces prolonged formation, in a Ras-dependent manner, of transcriptionally active NF-kappaB DNA-binding complexes, which make up the family of Rel-related proteins, p50, p52, RelA, and c-Rel. HBx was found to activate NF-kappaB through two distinct cytoplasmic pathways by acting on both the 37-kDa IkappaBalpha inhibitor and the 105-kappaDa NF-kappaB1 precursor inhibitor protein, known as p105. HBx induces phosphorylation of IkappaBalpha, a three- to fourfold reduction in IKappaBalpha stability, and concomitant nuclear accumulation of NF-kappaB DNA-binding complexes, similar to that reported for human T-cell leukemia virus type 1 Tax protein. In addition, HBx mediates a striking reduction in cytoplasmic p105 NF-kappaB1 inhibitor and p50 protein levels and release of RelA protein that was sequestered by the p105 inhibitor, concomitant with nuclear accumulation of NF-kappaB complexes. HBx mediated only a slight reduction in the cytoplasmic levels of NF-kappaB2 p100 protein, an additional precursor inhibitor of NF-kappaB, which is thought to be less efficiently processed or less responsive to release of NF-kappaB. No evidence was found for HBx activation of NF-kappaB by targeting acidic sphingomyelinase- controlled pathways. Studies also suggest that stimulation of NF-kappaB by HBx does not involve activation of Ras via the neutral sphingomyelin-ceramide pathway. Thus, HBx protein is shown to activate the NF-kappaB family of Rel-related proteins by acting on two distinct NF-kappaB cytoplasmic inhibitors (Su, 1996).

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

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