cactus

EVOLUTIONARY HOMOLOGS

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IkappaB domain structure and multiple forms

In vivo IkappaB alpha is a stronger inhibitor of NF-kappaB than is IkappaB beta. This difference is directly correlated with their varying abilities to inhibit NF-kappaB binding to DNA in vitro and in vivo. Moreover, IkappaB alpha, but not IkappaB beta, can remove NF-kappaB from functional preinitiation complexes in in vitro transcription experiments. Both IkappaBs function in vivo not only in the cytoplasm but also in the nucleus, where they inhibit NF-kappaB binding to DNA. The inhibitory activity of IkappaB beta, but not that of IkappaB alpha, is facilitated by phosphorylation of the C-terminal PEST sequence by casein kinase II and/or by the interaction of NF-kappaB with high-mobility group protein I (HMG I) on selected promoters. The unphosphorylated form of IkappaB beta forms stable ternary complexes with NF-kappaB on the DNA either in vitro or in vivo. These experiments suggest that IkappaB alpha works as a postinduction repressor of NF-kappaB independently of HMG I, whereas IkappaB beta functions preferentially in promoters regulated by the NF-kappaB/HMG I complexes (Tran, 1997).

The Rel/NF-kappaB family of transcription factors controls the expression of a wide variety of genes that are implicated in immune and inflammatory responses and cellular proliferation. Disregulation of NF-kappaB is associated with cellular transformation and the maintenance of a high anti-apoptotic threshold in transformed cells. NF-kappaB activity is in turn regulated by its sequestration in the cytoplasm by the inhibitor I kappaB. I kappaB alpha, the most abundant and well-characterized member of the I kappaB multiprotein family, is rapidly degraded in response to multiple physiologic stimuli. Not only the amino-terminus, but also the carboxy-terminus of I kappaB alpha contains transferable signals that must be simultaneously present in an unrelated protein to render it susceptible to activation-induced, proteasome-mediated degradation. I kappaB alpha amino-terminal modifications occur independently of the carboxy-terminus. The carboxy-terminal region has a critical role in facilitating proteolysis by the catalytic core of the proteasome. When incubated with 20S proteasome extracted from rat liver, I kappaB alpha is quickly degraded; in contrast, a deletion mutant lacking the carboxy-terminus is resistant to proteolysis. Likewise, chimeric proteins of beta-galactosidase with the I kappaB alpha carboxy-terminus are degraded in vitro, independent of the presence of the I kappaB alpha amino-terminus, whereas chimeric proteins lacking the I kappaB alpha carboxy-terminus are stable. These results identify the carboxy-terminus of I kappaB alpha as a domain critical for degradation through interaction with an as yet unidentified component of the proteasome (Kroll, 1997).

IkappaBalpha is rapidly phosphorylated and degraded in response to stimulation through tumor necrosis factor alpha (TNFalpha) receptor, interleukin-1 receptor (a mammalian homolog of Toll) or CD40. To explore the molecular mechanisms of signal-induced depletion of IkappaBalpha, the domain in IkappaBalpha has been delineated that is required for TNFalpha-induced phosphorylation and rapid degradation of IkappaBalpha. In contrast to previous reports, the PEST-like sequences, which are present in the carboxyl-terminal region of IkappaBalpha, are shown to be dispensable for TNFalpha-induced degradation but could be required for signal-independent degradation, as in the case of Cactus, the Drosophila homolog of IkappaB. The ankyrin repeats, which are essential for forming a complex with Rel and RelA, are required for TNFalpha-induced degradation, suggesting that the putative IkappaB protease could interact with IkappaBalpha in complex with RelA or could recognize the structure of ankyrin repeats. These data also indicate that neither the ankyrin repeats nor the PEST-like sequences, are essential for TNFalpha-induced phosphorylation (Aoki, 1996).

The mRNA for a novel gene from a human epithelial cell line was detected only in the heart and the skeletal muscle. The gene, named I kappa BR (for I kappa B-related), has a 52-kDa protein product and show significant homology to the I kappa B family of proteins. The deduced amino acid sequence of I kappa BR has the most significant homology to the Drosophila protein Cactus, which inhibits the function of the NF-kappa B-like protein Dorsal. In transfection experiments, overexpression of I kappa BR significantly inhibits NF-kappa B-dependent transcription from the Ig kappa enhancer. This new member of the I kappa B family of proteins, I kappa BR, may play an important role in regulation of NF-kappa B function in epithelial cells (Ray, 1995).

To release transcription factor NF-kappaB into the nucleus, the mammalian IkappaB molecules IkappaB alpha and IkappaB beta are inactivated by phosphorylation and proteolytic degradation. Both proteins contain conserved signal-responsive phosphorylation sites and have conserved ankyrin repeats. To confer specific physiological functions to members of the NF-kappaB/Rel family, the different IkappaB molecules could vary in their specific NF-kappaB/Rel factor binding activities and could respond differently to activation signals. Both mechanisms apply to differential regulation of NF-kappaB function by IkappaB beta relative to IkappaB alpha. Via alternative RNA processing, human IkappaB beta gives rise to different protein isoforms. IkappaB beta1 and IkappaB beta2, the major forms in human cells, differ in their carboxy-terminal PEST sequences. IkappaB beta2 is the most abundant species in a number of human cell lines tested, whereas IkappaB beta1 is the only form detected in murine cells. These isoforms are indistinguishable in their preference for binding to cellular NF-kappaB/Rel homo- and hetero-dimers. Both isoforms are constitutively phosphorylated. However, in unstimulated B cells, IkappaB beta1 (but not IkappaB beta2) is found in the nucleus. The two forms differ markedly in their efficiency of proteolytic degradation after stimulation with several inducing agents tested. While IkappaB beta1 is nearly as responsive as IkappaB alpha, indicating a shared activation mechanism, IkappaB beta2 is only weakly degraded and often not responsive at all. Alternative splicing of the IkappaB beta pre-mRNA may thus provide a means to selectively control the amount of IkappaB beta-bound NF-kappaB heteromers to be released under NF-kappaB stimulating conditions (Hirano, 1998).

The biological activity of the transcription factor NF-kappaB is differentially controlled by three IkappaB proteins: IkappaBalpha, IkappaBbeta, and IkappaBepsilon. The molecular basis for the differential inhibitory strengths of IkappaB proteins has been examined by constructing hybrid IkappaBs. The first ankyrin repeat of IkappaBalpha is responsible for its strong inhibitory effect. Swapping a putative beta-turn within the first ankyrin repeat between the strong IkappaBalpha and the weak IkappaBbeta inhibitors switches their in vivo inhibitory activity on NF-kappaB. The qualitatively distinct contacts made by this beta-turn in IkappaBalpha and IkappaBbeta with NF-kappaB determine the efficiency by which IkappaBs sequester NF-kappaB to the cytoplasm, thus explaining their distinct effects on gene activity (Simeonidis, 1999).

Interaction of IkappaB with NF-kappaB

Activation of transcription factor NF-kappaB involves the signal-dependent degradation of basally phosphorylated inhibitors such as IkappaBalpha and IkappaBbeta. The gene encoding IkappaBalpha is under NF-kappaB control, which provides a negative feedback loop to terminate the induced NF-kappaB response. However, recent studies have identified a hypophosphorylated pool of IkappaBbeta that shields nuclear NF-kappaB from inhibition by newly synthesized IkappaBalpha. This protection mechanism is regulated by the C-terminal PEST domain of IkappaBbeta. Disruption of two basal phosphoacceptors present in the IkappaBbeta PEST domain (Ser-313 and Ser-315) yields a mutant that forms ternary complexes with NF-kappaB and its target DNA-binding site. Based on in vitro mixing experiments, these ternary complexes are resistant to the inhibitory action of IkappaBalpha. Mutants of IkappaBbeta that are defective for phosphorylation at Ser-313 and Ser-315 fail to efficiently block NF-kappaB-directed transcription in vivo, whereas replacement of these two IkappaBbeta residues with a phosphoserine mimetic generates a fully functional repressor. Taken together, these findings suggest that the functional fate of NF-kappaB when bound to IkappaBbeta is critically dependent on the phosphorylation status of the IkappaBbeta PEST domain (McKinsey, 1997).

IkappaBalpha is a critical regulator of Rel/NF-KB-mediated gene activation. It controls the induction of NF-KB factors by retaining them in the cytoplasm and also functions in the nucleus to terminate the induction process. IkappaBalpha regulates the transcriptional activity of c-Rel in the nuclear compartment. Discrete functional domains of IkappaBalpha are responsible for the cytoplasmic and nuclear regulation of c-Rel. The determinants for the cytoplasmic regulation of c-Rel reside in the N-terminal and central ankyrin regions of IkappaBalpha; the N-terminal domain of IkappaBalpha is required to mask the c-Rel nuclear localization signal. Importantly, IkappaBalpha sequences that are necessary to regulate c-Rel in the nucleus, map to its central ankyrin domain and to a few negatively charged amino acids that immediately follow in the C-terminal IkappaBalpha PEST domain. The mapping of the IkappaBalpha determinants that control the cytoplasmic and nuclear activities of c-Rel to specific regions of the molecule suggests that IkappaBalpha inhibitors could be designed to antagonize Rel/NF-kappaB activity in different subcellular compartments or at defined stages of activation (Luque, 1998).

Nuclear factor kappa B (NF-kappa B), a regulator of immune system and inflammation genes, may be a target for glucocorticoid-mediated immunosuppression. The activation of NF-kappa B involves the targeted degradation of its cytoplasmic inhibitor, I kappa B alpha, and the translocation of NF-kappa B to the nucleus. The synthetic glucocorticoid dexamethasone induces the transcription of the I kappa B alpha gene, which results in an increased rate of I kappa B alpha protein synthesis. Stimulation by tumor necrosis factor causes the release of NF-kappa B from I kappa B alpha. However, in the presence of dexamethasone this newly released NF-kappa B quickly reassociates with newly synthesized I kappa B alpha, thus markedly reducing the amount of NF-kappa B that translocates to the nucleus. This decrease in nuclear NF-kappa B is predicted to markedly decrease cytokine secretion and thus effectively block the activation of the immune system (Scheinman, 1995).

The transcription factor NF-kappaB is normally sequestered in the cytoplasm by members of the IkappaB family, including IkappaB alpha, IkappaB beta, and the recently cloned IkappaB epsilon. Upon cellular activation, these inhibitors are rapidly phosphorylated on two amino-terminal serines, then ubiquitinated, and degraded by the 26S proteasome, releasing a functional NF-kappaB. To determine the importance of IkappaB beta in NF-kappaB regulation in T cells, transgenic mice were generated expressing a constitutively active IkappaB beta mutant (mIkappaB beta) under the control of the lck promoter. The transgene contains the two critical N-terminal serine residues mutated to alanines and therefore no longer susceptible to degradation upon cell activation. mIkappaB beta is unable to totally displace IkappaB alpha from RelA-containing complexes, thus allowing a transient activation of NF-kappaB upon T-cell stimulation. However, mIkappaB beta completely blocks NF-kappaB activity after IkappaB alpha degradation. As a consequence of this inhibition, ikappaba expression is down-regulated, along with that of other NF-kappaB-regulated genes. These transgenic mice have a significant reduction in the peripheral T-cell population, especially CD8+ cells. The remaining T cells have impaired proliferation in response to phorbol 12-myristate 13-acetate plus phytohemagglutinin or calcium ionophore but not to anti-CD3/anti-CD28 costimulation. As a result of these alterations, transgenic animals present defects in immune responses, such as delayed-type hypersensitivity and the generation of specific antibodies against T-cell-dependent antigens. These results show that in nonstimulated T cells, IkappaB beta cannot efficiently displace IkappaB alpha bound to RelA-containing complexes and that persistent NF-kappaB activity is required for proper T-cell responses in vivo (Attar, 1998).

Members of the Rel/NF-kappaB family of transcription factors are related to each other over a region of about 300 amino acids called the Rel Homology Domain (RHD), which governs DNA binding, dimerization, and binding to inhibitor. At the C-terminal end of the RHD, each protein has a nuclear localization signal (NLS). The crystal structures of the p50 and RelA family members show that the RHD consists of two regions: an N-terminal section that contains some of the DNA contacts and a C-terminal section that contains the remaining DNA contacts and which controls dimerization. In unstimulated cells, the homo- or heterodimeric Rel/NF-kappaB proteins are cytoplasmic by virtue of binding to an inhibitor protein (IkappaB), which somehow masks the NLS of each member of the dimer. The IkappaB proteins consist of an ankyrin-repeat-containing domain that is required for binding to dimers and N- and C-terminal domains that are dispensable for binding to most dimers. The interaction between IkappaB alpha and Rel family homodimers has been examined by mutational analysis. The dimerization regions of p50, RelA, and c-Rel are sufficient for binding to IkappaB alpha. It is shown that the NLSs of RelA and c-Rel are not required for binding to IkappaB alpha but do stabilize the interaction, while the NLS of p50 is required for binding to IkappaB alpha. Only certain residues within the p50 NLS are required for binding. In a p50-IkappaB alpha complex or a c-Rel-IkappaB alpha complex, the N terminus of IkappaB alpha either directly or indirectly masks one or both of the dimer NLSs (Latimer, 1998).

Several lines of evidence have led to the suggestion that newly synthesized IkappaB can function in the nucleus as a postinduction repressor of B-dependent gene expression.

(1) Ectopically overexpressed IkappaB is readily detected in the nucleus, consistent with the suggestion that IkappaB has a nuclear function.
(2) Following cytokine stimulation of cells, a significant fraction of newly synthesized endogenous IkappaB appears transiently in the nucleus. Nuclear expression of IkappaB correlates with inhibition of NFkappa-B-dependent transcription and disappearance of NFkappa-B from the nucleus.
(3) Brief stimulation of wild-type fibroblasts with tumor necrosis factor alpha (TNF-alpha) results in a transient activation of nuclear NF-kappaB. In contrast, brief stimulation of IB null-mutant fibroblasts with TNF-alpha results in a sustained level of nuclear NFkappa-B.
(4) IkappaB can inhibit NFkappa-B-dependent transcription in the nucleus in vivo and can remove Rel proteins from functional preinitiation complexes in vitro.

Taken together, these results are consistent with a model in which newly synthesized IkappaB proteins can enter the nucleus, displace dimeric Rel proteins from DNA, and export Rel proteins from the nucleus to the cytoplasm. Implicit in this model is the ability of both Rel and IkappaB proteins to enter the nucleus. In contrast, this model postulates that the Rel-IkappaB complex is exported from the nucleus and is efficiently retained in the cytoplasm (Sachdev, 1998a).

The IkappaB alpha protein is able both to inhibit nuclear import of Rel/NF-kappaB proteins and to mediate the export of Rel/NF-kappaB proteins from the nucleus. The c-Rel-IkappaB alpha complex is stably retained in the cytoplasm in the presence of leptomycin B, a specific inhibitor of Crm1-mediated nuclear export. In contrast, leptomycin B treatment results in the rapid and complete relocalization of the v-Rel-IkappaB alpha complex from the cytoplasm to the nucleus. IkappaB alpha also mediates the rapid nuclear shuttling of v-Rel in an interspecies heterokaryon assay. Thus, continuous nuclear export is required for cytoplasmic retention of the v-Rel-IkappaB alpha complex. Furthermore, although IkappaB alpha is able to mask the c-Rel-derived nuclear localization sequence (NLS), IkappaB alpha is unable to mask the v-Rel-derived NLS in the context of the v-Rel-IkappaB alpha complex. Taken together, these results demonstrate that IkappaB alpha is unable to inhibit nuclear import of v-Rel. Two amino acid differences between c-Rel and v-Rel (Y286S and L302P) that link to oncogenesis have been identified: (1) the failure of IkappaB alpha to inhibit nuclear import and (2) DNA binding of a mutant c-Rel protein. These results support a model in which loss of IkappaB alpha-mediated control over c-Rel leads to oncogenic activation of c-Rel (Sachdev, 1998b).

The ability of the IkappaB alpha protein to sequester dimeric NF-kappaB/Rel proteins in the cytoplasm provides an effective mechanism for regulating the potent transcriptional activation properties of NF-kappaB/Rel family members. IkappaB alpha can also act in the nucleus as a postinduction repressor of NF-kappaB/Rel proteins. The mechanism by which IkappaB alpha enters the nucleus is not known, as IkappaB alpha lacks a discernible classical nuclear localization sequence (NLS). Nuclear localization of IkappaB alpha is mediated by a novel nuclear import sequence within the second ankyrin repeat. Deletion of the second ankyrin repeat or alanine substitution of hydrophobic residues within the second ankyrin repeat disrupts nuclear localization of IkappaB alpha. A region encompassing the second ankyrin repeat of IkappaB alpha is able to function as a discrete nuclear import sequence. The presence of a discrete nuclear import sequence in IkappaB alpha suggests that cytoplasmic sequestration of the NF-kappaB/Rel-IkappaB alpha complex is a consequence of the mutual masking of the NLS within NF-kappaB/Rel proteins and the import sequence within IkappaB alpha. Nuclear import may be a conserved property of ankyrin repeat domains (ARDs), because the ARDs from two other ARD-containing proteins, 53BP2 and GABPbeta, are also able to function as nuclear import sequences. It is proposed that the IkappaB alpha ankyrin repeats define a novel class of cis-acting nuclear import sequences (Sachdev, 1998).

IkappaBalpha regulates the transcription factor NF-kappaB through the formation of stable IkappaBalpha/NF-kappaB complexes. Prior to induction, IkappaBalpha retains NF-kappaB in the cytoplasm until the NF-kappaB activation signal is received. After activation, NF-kappaB is removed from gene promoters through association with nuclear IkappaBalpha, restoring the preinduction state. The 2.3 A crystal structure of IkappaBalpha in complex with the NF-kappaB p50/p65 heterodimer reveals mechanisms of these inhibitory activities. The presence of IkappaBalpha allows large en bloc movement of the NF-kappaB p65 subunit amino-terminal domain. This conformational change induces allosteric inhibition of NF-kappaB DNA binding. Amino acid residues immediately preceding the nuclear localization signals of both NF-kappaB p50 and p65 subunits are tethered to the IkappaBalpha amino-terminal ankyrin repeats, impeding NF-kappaB from nuclear import machinery recognition (Huxford, 1998).

The inhibitory protein, IkappaBalpha, sequesters the transcription factor, NF-kappaB, as an inactive complex in the cytoplasm. The structure of the IkappaBalpha ankyrin repeat domain, bound to a partially truncated NF-kappaB heterodimer (p50/ p65), has been determined by X-ray crystallography at 2.7 A resolution. It shows a stack of six IkappaBalpha ankyrin repeats facing the C-terminal domains of the NF-kappaB Rel homology regions. Contacts occur in discontinuous patches, suggesting a combinatorial quality for ankyrin repeat specificity. The first two repeats cover an alpha helically ordered segment containing the p65 nuclear localization signal. The position of the sixth ankyrin repeat shows that full-length IkappaBalpha will occlude the NF-kappaB DNA-binding cleft. The orientation of IkappaBalpha in the complex places its N- and C-terminal regions in appropriate locations for their known regulatory functions (Jacobs, 1998).

Negative selection eliminates thymocytes bearing autoreactive T cell receptors (TCR) via an apoptotic mechanism. An inhibitor of NF-kappaB, IBNS, has been cloned, that is rapidly expressed upon TCR-triggered but not dexamethasone- or irradiation-stimulated thymocyte death. The predicted protein contains seven ankyrin repeats and is homologous to IkappaB family members. In class I and class II MHC-restricted TCR transgenic mice, transcription of IBNS is stimulated by peptides that trigger negative selection but not by those inducing positive selection (i.e., survival) or nonselecting peptides. IBNS blocks transcription from NF-kappaB reporters, alters NF-kappaB electrophoretic mobility shifts, and interacts with NF-kappaB proteins in thymic nuclear lysates following TCR stimulation. Retroviral transduction of IBNS in fetal thymic organ culture enhances TCR-triggered cell death consistent with its function in selection (Fiorini, 2002).

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