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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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