Frank Mercurio

NF-κB and the link between inflammation and cancer

Summary:  The nuclear factor‐κB (NF‐κB) transcription factor family has been considered the central mediator of the inflammatory process and a key participant in innate and adaptive immune responses. Coincident with the molecular cloning of NF‐κB/RelA and identification of its kinship to the v‐Rel oncogene, it was anticipated that NF‐κB itself would be involved in cancer development. Oncogenic activating mutations in NF‐κB genes are rare and have been identified only in some lymphoid malignancies, while most NF‐κB activating mutations in lymphoid malignancies occur in upstream signaling components that feed into NF‐κB. NF‐κB activation is also prevalent in carcinomas, in which NF‐κB activation is mainly driven by inflammatory cytokines within the tumor microenvironment. Importantly, however, in all malignancies, NF‐κB acts in a cell type‐specific manner: activating survival genes within cancer cells and inflammation‐promoting genes in components of the tumor microenvironment. Yet, the complex biological functions of NF‐κB have made its therapeutic targeting a challenge.

Multiple signals converging on NF-κB

Abstract The recent identification of molecular components of the signal transduction pathway regulating activation of nuclear factor-κB (NF-κB) in response to cytokines such as tumor necrosis factor α and interleukin-1β allows the evaluation of how other diverse stimuli impinge on the NF-κB activation pathway. These studies suggest a basis for specificity in activation of specific Rel-related family members and the genetic responses they promote.

Identification of the receptor component of the IκBα-ubiquitin ligase

NF-κB, 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, IκBs. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin–proteasome system to eliminate inhibitory regulators. Here we isolate the pIκBα–ubiquitin ligase (pIκBα-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-κB inhibitor pIκBα. Taking advantage of its high affinity to pIκBα, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIκBα degradation motif as an F-box/WD-domainprotein belonging to a recently distinguished family of β-TrCP/Slimb proteins. This component, which we denote E3RSIκB (pIκBα-E3 receptor subunit), binds specifically to pIκBα 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 E3RSIκB, which tightly binds pIκBα but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIκBα and consequently NF-κB activation. E3RSIκB 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.

NF-κB as a primary regulator of the stress response

A myriad of unrelated exogenous or endogenous agents that represent a threat to the organism are capable of inducing NF-κB activity, including viral infection, bacterial lipids, DNA damage, oxidative stress and chemotherapuetic agents. Likewise, NF-κB regulates the expression of an equally diverse array of cellular genes. These findings are indicative of the widespread significance of NF-κB as a mediator of cellular stress. Remarkably, the NF-κB pathway displays the capacity to activate, in a cell- and stimulus-specific manner, only a subset of the total repertoire of NF-κB-responsive genes. The seemingly promiscuous nature of NF-κB activation poses a regulatory quagmire as to how specificity is achieved at the level of gene expression. The review will summarize recent findings and explore how they further our understanding of the mechanism by which stimulus-specific activation of NF-κB is achieved in response to cellular stress.

IκB Kinase (IKK)-Associated Protein 1, a Common Component of the Heterogeneous IKK Complex

ABSTRACT Activation of the transcription factor NF-κB is controlled by the sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit, IκB. We recently purified a large multiprotein complex, the IκB kinase (IKK) signalsome, which contains two regulated IκB kinases, IKK1 and IKK2, that can each phosphorylate IκBα and IκBβ. The IKK signalsome contains several additional proteins presumably required for the regulation of the NFκB signal transduction cascade in vivo. In this report, we demonstrate reconstitution of IκB kinase activity 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 we identified two distinct IKK complexes, one containing IKK1-IKK2 heterodimers and the other containing IKK2 homodimers, which display differing levels of activation following tumor necrosis factor alpha stimulation. To better elucidate the nature of the IKK signalsome, we set out to identify IKK-associated proteins. To this end, we purified and cloned a novel component common to both complexes, named IKK-associated protein 1 (IKKAP1). In vitro, IKKAP1 associated specifically with IKK2 but not IKK1. Functional analyses revealed 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-κB signal transduction pathway. IKKAP1 therefore appears to mediate an essential step of the NF-κB signal transduction cascade. Heterogeneity of IKK complexes in vivo may provide a mechanism for differential regulation of NF-κB activation.

Identification of the receptor component of the IkappaBalpha-ubiquitin ligase.

NF-κB, 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, IκBs. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin–proteasome system to eliminate inhibitory regulators. Here we isolate the pIκBα–ubiquitin ligase (pIκBα-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-κB inhibitor pIκBα. Taking advantage of its high affinity to pIκBα, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIκBα degradation motif as an F-box/WD-domainprotein belonging to a recently distinguished family of β-TrCP/Slimb proteins. This component, which we denote E3RSIκB (pIκBα-E3 receptor subunit), binds specifically to pIκBα 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 E3RSIκB, which tightly binds pIκBα but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIκBα and consequently NF-κB activation. E3RSIκB 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.

HTLV-I Tax Protein Binds to MEKK1 to Stimulate IκB Kinase Activity and NF-κB Activation

Abstract NF-κB, a key regulator of the cellular inflammatory and immune response, is activated by the HTLV-I transforming and transactivating protein Tax. We show that Tax binds to the amino terminus of the protein kinase MEKK1, a component of an IκB kinase complex, and stimulates MEKK1 kinase activity. Tax expression increases the activity of IκB kinase β (IKKβ) to enhance phosphorylation of serine residues in IκBα that lead to its degradation. Dominant negative mutants of both IKKβ and MEKK1 prevent Tax activation of the NF-κB pathway. Furthermore, recombinant MEKK1 stimulates IKKβ phosphorylation of IκBα. Thus, Tax-mediated increases in NF-κB nuclear translocation result from direct interactions of Tax and MEKK1 leading to enhanced IKKβ phosphorylation of IκBα.

Inhibition of NF‐κB cellular function via specific targeting of the IκB‐ubiquitin ligase

Activation of the transcription factor NF‐κB is a paradigm for signal transduction through the ubiquitin–proteasome pathway: ubiquitin‐dependent degradation of the transcriptional inhibitor IκB 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. Here we show that following a stimulus‐dependent phosphorylation, and while associated with NF‐κB, IκB 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 IκB and its subsequent degradation. The ligase recognition signal is functionally conserved between IκBα and IκBβ, and does not involve the nearby ubiquitination site. Microinjection of the inhibitory peptides into stimulated cells abolished NF‐κB activation in response to TNFα and the consequent expression of E‐selectin, an NF‐κB‐dependent cell‐adhesion molecule. Inhibition of NF‐κB function by specific blocking of ubiquitin ligase activity provides a novel approach for intervening in cellular processes via regulation of unique proteolytic events.

Role of IKK1 and IKK2 in lipopolysaccharide signaling in human monocytic cells

Mononuclear phagocytes play a major role in immune and inflammatory responses. Bacterial lipopolysaccharide (LPS) induces monocytes to express a variety of genes by activating the NF-κB/Rel transcription factor family. Recently, we have reported that the tumor necrosis factor and interleukin 1 signaling pathways activate two kinases, IKK1 and IKK2. Phosphorylation of the IκB cytoplasmic inhibitors, IκBα, IκBβ, and IκBε, by these kinases triggers proteolytic degradation and the release of NF-κB/Rel proteins into the nucleus. At present, the role of the IKKs in LPS signaling has not been investigated. Here, we report that LPS induces IKK activity in human monocytes and THP-1 monocytic cells. The kinetics of activation of kinase activity in monocytic cells are relatively slow with maximal activity observed at 60 min, which coincides with the degradation of IκBs and the nuclear translocation of NF-κB. In transfection experiments, overexpression of wild type IKK1, a dominant negative mutant IKK1 (K44M), or wild type IKK2 did not affect LPS-induced κB-dependent transcription in monocytic cells. In contrast, a dominant negative mutant of IKK2 inhibited LPS induction of κB-dependent transcription in a dose-dependent manner. These results indicate that LPS induction of κB-dependent gene expression in human monocytic cells requires activation of IKK2.

SCFβ-TrCP ubiquitin ligase-mediated processing of NF-κB p 105 requires phosphorylation of its C-terminus by IκB kinase

Processing of the p105 precursor to form the active subunit p50 of the NF‐κB transcription factor is a unique case in which the ubiquitin system is involved in limited processing rather than in complete destruction of the target substrate. A glycine‐rich region along with a downstream acidic domain have been demonstrated to be essential for processing. Here we demonstrate that following IκB kinase (IκK)‐mediated phosphorylation, the C‐terminal domain of p105 (residues 918–934) serves as a recognition motif for the SCFβ‐TrCP ubiquitin ligase. Expression of IκKβ dramatically increases processing of wild‐type p105, but not of p105‐Δ918–934. Dominant‐negative β‐TrCP inhibits IκK‐dependent processing. Furthermore, the ligase and wild‐type p105 but not p105‐Δ918–934 associate physically following phosphorylation. In vitro, SCFβ‐TrCP specifically conjugates and promotes processing of phosphorylated p105. Importantly, the TrCP recognition motif in p105 is different from that described for IκBs, β‐catenin and human immunodeficiency virus type 1 Vpu. Since p105‐Δ918–934 is also conjugated and processed, it appears that p105 can be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs.