Among the several signalling pathways regulated by ubiquitin and ubiquitin-like proteins, the one activating NF-κB (nuclear factor κB) is certainly one of the best characterized. The regulation of the activity of this transcription factor by members of the ubiquitin family occurs at various levels, imposing overlapping controls of security of intriguing complexity. The formation of active macromolecular complexes such as the IKK [IκB (inhibitory κB) kinase] complex is tightly regulated by these post-translational modifications probably due to the fact that many signals converge on this signal's roundabout. An additional, very important level of NF-κB control occurs through the partial or total proteolysis of precursor and inhibitor molecules exerted by the ubiquitin–proteasome pathway. Regulation at this level implicates various conjugating and de-conjugating activities for ubiquitin, SUMO (small ubiquitin-related modifier) and NEDD8. Here, we summarize some of these events and underline the importance of the interconnecting ubiquitin and ubiquitin-like conjugating pathways that determine the status of the activity of this critical transcription factor.

Introduction

The NF-κB (nuclear factor κB) pathway is the conduit for signals leading to a variety of cellular responses, including the induction of pro-inflammatory and anti-apoptotic genes (reviewed by Perkins and Gilmore [1]). Numerous effector molecules are involved in a framework describing how the signal is generated from the receptor on the cell surface, transduced to the cytoplasm where the IKK [IκB (inhibitory κB) kinase] complex phosphorylates the inhibitory proteins (IκB), until the final release of the NF-κB transcription factor into the nucleus. Since the NF-κB pathway is involved in many cellular and biological functions and its deregulation has catastrophic consequences (cancer, autoimmune disease, inflammation etc.), its activity needs to be specifically and highly regulated. A major roundabout of the signalling cascades activating this transcription factor is certainly the IKK complex. The regulation of the IKK activity involves very complex ubiquitin-mediated proteolytic and non-proteolytic events that are still under characterization. In mammals, NF-κB is a heterodimeric transcription factor composed of a transcriptionally active subunit that could be RelA (p65), RelB and c-Rel and a transcriptionally dead subunit: p50 or p52. The most commonly expressed NF-κB dimer is composed of p65/p50 [2]. The activity of NF-κB is conditioned by its interaction with the natural IκB inhibitors, which retain it in the cytoplasm in an inactive form by masking its nuclear localization. Therefore the activation of NF-κB requires the destruction of IκB molecules to release and locate NF-κB into the nucleus where it will play its role as a transcription factor. In addition to the cytoplasmic retention, newly synthesized IκBα (generated after a first round of transcription activation) is also able to promote the nuclear export of promoter-bound NF-κB ensuring the termination of its activity [3,4].

Regulation of NF-κB by ubiquitin-mediated proteolysis

There are several forms of IκB, which possess distinctive tissue/functional characteristics; among them, IκBα, IκBβ and IκBε are the best characterized. In addition to the characteristic centric ankyrin repeats, they all contain the IKK phosphorylation consensus DSG (X)2+nS that triggers the ubiquitin-mediated degradation of these molecules. This signal is recognized by the ubiquitin ligase SCF (Skp1/cullin/F-box)-β-TrcP (β-transducin repeat-containing protein) that together with the E2s Ubc5 or Cdc34 (cell division cycle 34) allows the ubiquitination of these inhibitors. In the case of the IκBα, ubiquitination occurs on lysine residues Lys-21 and Lys-22, resulting in its proteasomal-mediated degradation [5]. Ubiquitin E3s of the SCF type integrate a protein module [RBX (RING box)] containing the RING (really interesting new gene) finger structure responsible for the interaction with (i) the E2, (ii) a scaffold-like cullin molecule, (iii) the protein adaptor SKP1 and (iv) an F-box-containing protein, which recognize the substrate [6]. An additional level of complexity concerns the regulation of the activity of SCF ligases by conjugation/de-conjugation of the ubiquitin-like modifier NEDD8. Neddylation conditions the association of cullins with RBX proteins [7]. In this process, CAND1 (cullin-associated and neddylation associated protein 1) plays a regulatory role by interfering with the neddylation of cullins and therefore the association with the rest of the subunits required for generating a functional SCF complex. While neddylation involves Ubc12 and APP-BP1 [APP (amyloid precursor protein)-binding protein-1]/UBA3 (ubiquitin-like modifier activating enzyme 3) enzymes, de-neddylation is mediated by a subunit of the CSN (COP9 signalosome) complex. The assembly/disassembly of cullin-based E3s appears to condition the in vivo activity of these enzymes [8] (Figure 1).

Interacting ubiquitin and ubiquitin-like conjugating/de-conjugating pathways regulate IκBα modification

Figure 1
Interacting ubiquitin and ubiquitin-like conjugating/de-conjugating pathways regulate IκBα modification

The UCH (ubiquitin C-terminal hydrolase) generates mature ubiquitin. Ubiquitin is activated by the E1 activating enzyme. The E2s Ubc5 or Cdc34 together with the SCF-β-TrcP ligase have been involved in the conjugation of ubiquitin to IκBα. Neddylation and de-neddylation of one of the components of the SCF are required for the full activity of the SCF-β-TrcP ligase. UCHL3 and DEN1 have been involved in the generation of the mature form of NEDD8 through an NCH (NEDD8 C-terminal hydrolase) activity. To activate NEDD8, the APP-BP1–UBA3 dimer works as an E1. Together with a RING finger protein (Rbx/Roc) of the SFC complex, the E2 Ubc12 conjugates NEDD8 to the cullin-1 subunit. To generate fully active SCF-β-TrcP, the action of the de-neddylating subunit of the CSN is required. The action of a UBP (unidentified ubiquitin protease) removing ubiquitin from the Lys-21 will facilitate SUMOylation of IκBα. This last modification requires inactivation of the SEA (SUMO-activating enzyme) and Ubc9. The necessity of a SUMO E3 for the SUMOylation of this NF-κB inhibitor has not been demonstrated. The action of one or more SUSP (SUMO-specific proteases) might be certainly critical for the maturation and de-conjugation of SUMO molecules regulating IκBα activity.

Figure 1
Interacting ubiquitin and ubiquitin-like conjugating/de-conjugating pathways regulate IκBα modification

The UCH (ubiquitin C-terminal hydrolase) generates mature ubiquitin. Ubiquitin is activated by the E1 activating enzyme. The E2s Ubc5 or Cdc34 together with the SCF-β-TrcP ligase have been involved in the conjugation of ubiquitin to IκBα. Neddylation and de-neddylation of one of the components of the SCF are required for the full activity of the SCF-β-TrcP ligase. UCHL3 and DEN1 have been involved in the generation of the mature form of NEDD8 through an NCH (NEDD8 C-terminal hydrolase) activity. To activate NEDD8, the APP-BP1–UBA3 dimer works as an E1. Together with a RING finger protein (Rbx/Roc) of the SFC complex, the E2 Ubc12 conjugates NEDD8 to the cullin-1 subunit. To generate fully active SCF-β-TrcP, the action of the de-neddylating subunit of the CSN is required. The action of a UBP (unidentified ubiquitin protease) removing ubiquitin from the Lys-21 will facilitate SUMOylation of IκBα. This last modification requires inactivation of the SEA (SUMO-activating enzyme) and Ubc9. The necessity of a SUMO E3 for the SUMOylation of this NF-κB inhibitor has not been demonstrated. The action of one or more SUSP (SUMO-specific proteases) might be certainly critical for the maturation and de-conjugation of SUMO molecules regulating IκBα activity.

IκBα can also be modified on Lys-21 by SUMO-1 (small ubiquitin-related modifier-1) resulting in an inhibition of the NF-κB activity [9]. This lysine residue fits the SUMOylation consensus but requires the presence of an NLS (nuclear localization sequence) to be modified in vivo [10]. Because SUMOylation occurs on one of the two lysine residues modified by ubiquitination after cell stimulation with TNFα (tumour necrosis factor α) or IL-1β (interleukin-1β), SUMOylation of IκBα might interfere with the ubiquitination of this NF-κB inhibitor. Inversely, SUMOylation of IκBα appears to be inhibited by phosphorylation on the serine residues Ser-32/Ser-36 and is therefore negatively regulated by the signal-mediated ubiquitination [9] (Figure 1). Although this was the first model of competition between SUMO and ubiquitin for the modification of a protein substrate and several other examples have been published, many molecular aspects of this competition remain to be clarified [11,12]. Considering that other SUMO targets from the NF-κB signalling pathway have been reported since the IκBα SUMOylation studies, previous conclusions from SUMO-1 overexpression strategies might have to be reconsidered [13].

A simultaneous event activated after cell stimulation is the UPS (ubiquitin-26S proteasome)-mediated processing or degradation of the high-molecular-mass precursor p100 and p105 respectively. This processing generates the transcriptionally inactive subunits p50 and p52, which will integrate the various NF-κB heterodimers. Both precursors contain ankyrin repeats behaving as IκB-like proteins that are destroyed in a tissue- and stimuli-specific manner after activation of different pathways. While the processing of p100 required to generate p52 is induced by lymphotoxin β, B-cell activating factor or CD40 ligand, the processing of p105 appears to be constitutive. However, after stimulation with TNFα or lipopolysaccharides, p105 is degraded by the proteasome. In this event, the phosphorylation consensus recognized by the SCF-β-TrcP ubiquitin ligase is required to drive a ubiquitin-mediated degradation [14].

Regulation of NF-κB by ubiquitin and ubiquitin-like proteins

Indications of a proteasome-independent inductive role for ubiquitination upstream of IKKs were first provided by in vitro reconstitution experiments [15]. Surprisingly, it was shown that activation of the IKK complex requires ubiquitination, independently of the proteasome, and thus provided a first hint of a non-degradative role for ubiquitin in the NF-κB pathway. Since then, it has been demonstrated that the first step in IKK activation involves modification of the regulatory subunit of the IKK complex, NEMO (NF-κB essential modulator; or IKKγ), through Lys-63-linked polyubiquitination [1618]. While Lys-48-linked oligomers cause proteasomal degradation, Lys-63-linked chains usually serve to modify substrate activity. According to recent structural studies, it seems that ubiquitin chains linked by Lys-48 and Lys-63 adopt different configurations, potentially providing the molecular basis for the distinct signalling functions of these polyubiquitin chains [19]. In the case of NEMO, non-degradative ubiquitin chains seem to act as a scaffolding interface, which assembles activating kinases and effector kinases in close proximity. Alternatively, the critical step in IKK activation could be conformational changes that are introduced into the IKK complex by Lys-63 modification and perhaps attraction of ubiquitin-binding proteins. However, NEMO is just one component in a complex series of ubiquitin-based modifications of the signalling molecules involved in IKK activation. In fact, autoubiquitination of TRAF-2 [TNFR (TNF receptor)-associated factor-2] and TRAF-6 as well as ubiquitination of a scaffold protein like RIP1 (receptor-interacting protein 1) are also necessary for the IKK activation. Polyubiquitination of these signalling proteins allows targeting of IKK activating enzymes like TAK1 [TGF (transforming growth factor)-β-activated kinase 1] to the complex. TAK1 is associated with TAB2 and TAB3 that are themselves Lys-63-linked polyubiquitin-binding proteins (Figure 2).

Regulation of NF-κB by ubiquitin and ubiquitin-like proteins

Figure 2
Regulation of NF-κB by ubiquitin and ubiquitin-like proteins

Many stimuli induce the activation of NF-κB and even if the signalling molecules involved are different, all lead to the activation of the IKK complex. IKK will phosphorylate IκBα, which induces its association with the SCF complex, its polyubiquitination and subsequently its degradation by the proteasome. Stimulation of TNFR leads to the activation of ubiquitin ligase activities of TRAF-2, promoting the Lys-63 polyubiquitination of TRAF-2 itself but also of RIP1 in the presence of the Ubc13–Uev1 E2 complex. The polyubiquitinated RIP1 recruits the TAK1 kinase complex through the interaction between TAB2 and TAB3, but also NEMO, which will allow TAK1 to phosphorylate and activate IKK. The IL-1R (IL-1 receptor) and TLR signalling pathways activate IKK through TRAF-6, which functions as an E3 ligase as well as a target of Lys-63 polyubiquitination. The polyubiquitinated TRAF-6 recruits TAK1 and IKK complexes to mediate the activation of these kinases. In T-cells, the CBM complex induces the ligase activity of TRAF-6 and possibly MALT1 to catalyse the polyubiquitination of TRAF-6 itself and NEMO. These events are responsible for TAK1 and IKK activation and will ultimately induce Bcl-10 phosphorylation and its subsequent polyubiquitination and degradation by the proteasome. Two deubiquitinating enzymes are playing an important role in the regulation of NF-κB activity: CYLD and A20. Both can remove Lys-63 polyubiquitin chains from TRAFs and RIP1, thus suppressing the IKK activation. A20 can also function as an E3 ligase promoting the Lys-48 polyubiquitination of RIP1, which targets it for degradation by the proteasome. DNA damage leads to the SUMOylation and subsequent ubiquitination of NEMO in the nucleus. The ubiquitinated NEMO then exits the nucleus and associates with the other subunit of the IKK complex and induces its activity.

Figure 2
Regulation of NF-κB by ubiquitin and ubiquitin-like proteins

Many stimuli induce the activation of NF-κB and even if the signalling molecules involved are different, all lead to the activation of the IKK complex. IKK will phosphorylate IκBα, which induces its association with the SCF complex, its polyubiquitination and subsequently its degradation by the proteasome. Stimulation of TNFR leads to the activation of ubiquitin ligase activities of TRAF-2, promoting the Lys-63 polyubiquitination of TRAF-2 itself but also of RIP1 in the presence of the Ubc13–Uev1 E2 complex. The polyubiquitinated RIP1 recruits the TAK1 kinase complex through the interaction between TAB2 and TAB3, but also NEMO, which will allow TAK1 to phosphorylate and activate IKK. The IL-1R (IL-1 receptor) and TLR signalling pathways activate IKK through TRAF-6, which functions as an E3 ligase as well as a target of Lys-63 polyubiquitination. The polyubiquitinated TRAF-6 recruits TAK1 and IKK complexes to mediate the activation of these kinases. In T-cells, the CBM complex induces the ligase activity of TRAF-6 and possibly MALT1 to catalyse the polyubiquitination of TRAF-6 itself and NEMO. These events are responsible for TAK1 and IKK activation and will ultimately induce Bcl-10 phosphorylation and its subsequent polyubiquitination and degradation by the proteasome. Two deubiquitinating enzymes are playing an important role in the regulation of NF-κB activity: CYLD and A20. Both can remove Lys-63 polyubiquitin chains from TRAFs and RIP1, thus suppressing the IKK activation. A20 can also function as an E3 ligase promoting the Lys-48 polyubiquitination of RIP1, which targets it for degradation by the proteasome. DNA damage leads to the SUMOylation and subsequent ubiquitination of NEMO in the nucleus. The ubiquitinated NEMO then exits the nucleus and associates with the other subunit of the IKK complex and induces its activity.

TRAF molecules contain a RING finger domain, acting as E3 ligases and synthesizing Lys-63-linked chains. Oligomerization triggered upon ligand–receptor interaction induced E3 activity of TRAFs [20]. This process requires the ubiquitin-conjugating E2 enzyme Ubc13–Uev1A. While TRAF-6 ubiquitinates NEMO in the TLR (Toll-like receptor) and IL-1 pathways, TRAF-2 catalyses the ubiquitination of RIP1 in the TNFα pathway [21]. In TCR (T-cell receptor) and BCR (B-cell receptor) signalling, the CBM complex [CARMA1–Bcl-10–MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) complex] is essential for IKK and NF-κB activation. MALT1 seems to have a Lys-63-specific E3 activity, which is activated by Bcl-10-mediated oligomerization. As a consequence, MALT1 ubiquitinates NEMO. It has also been shown that Bcl-10 and MALT1 can induce the E3 activity of TRAF-6 [22].

This activation process is counteracted by deubiquitinating enzymes like A20 and CYLD (cylindromatosis protein), which can degrade Lys-63 chains. CYLD interferes with NF-κB signalling by catalysing the selective cleavage of Lys-63 chains from TRAF proteins and NEMO [2325]. A20 seems to down-regulate NF-κB by two mechanisms. A first step involves its N-terminal domain in the removal of Lys-63 chains from RIP and TRAF-6, and its C-terminal domain is involved in the ubiquitination and subsequent degradation of RIP1 by the proteasome [26,27].

A role of SUMO and SUMO enzymes, at different levels of the NF-κB pathway, has been suggested (for a review, see [13]). As mentioned above, SUMOylation of IκBα is involved in the regulation of NF-κB activity. Under certain conditions, the activity of the IKK complex seems to be controlled by SUMO modification. Indeed, it has been shown that a genotoxic stress induced the translocation of NEMO in the nucleus, where it will be modified by SUMO-1. In the nucleus, NEMO is subsequently de-SUMOylated and ubiquitinated at the same residues. Its ubiquitination requires its prior phosphorylation by ATM (ataxia telangiectasia mutated). Then, ubiquitinated NEMO is re-exported to the cytoplasm, where it can activate IKKα or IKKβ and induce the anti-apoptotic response of NF-κB [28].

Concluding remarks

For a while, polyubiquitin modification was thought to only target proteins for degradation by the proteasome, or to eliminate misfolded proteins or to terminate protein function. However, it is now known that ubiquitin can be attached to proteins in the form of multiple monomers or polymers of different topologies, and these topological variants lead to differences in function of the modified protein. An additional complexity came when it was realized that ubiquitin-like proteins, such as SUMO proteins or NEDD8, could modify similar proteins, generating agonistic or antagonistic effects to the ones regulated by ubiquitin conjugation. The complexity is even higher since it has been shown that SUMOylation of some substrates is necessary for their ubiquitination and degradation by the proteasome [29,30]. Moreover, de-conjugating enzymes for ubiquitin, NEDD8 and SUMO molecules have turned ubiquitin and ubiquitin-like proteins into highly dynamic modifiers whose attachments are tightly regulated both spatially and temporally. Therefore, to understand the role of these post-translational modifications, we have to keep in mind that these modifications are not necessarily independent events. After all the efforts made by the scientific community, it appears that we are just begining to understand the role of these modifications not only in the regulation of the NF-κB activity but also in all the events involving members of the ubiquitin family.

Third Intracellular Proteolysis Meeting: A joint Biochemical Society and INPROTEOLYS Network Focused Meeting held at Auditorio de Tenerife, Santa Cruz de Tenerife, Canary Islands, Spain, 5–7 March 2008. Organized and Edited by Rosa Farràs (Centro de Investigación Príncipe Felipe, Valencia, Spain), Gemma Marfany (Barcelona, Spain), Manuel Rodríguez (CICbioGUNE, Derio, Spain), Eduardo Salido (La Laguna, Tenerife, Spain) and Dimitris Xirodimas (Dundee, U.K.).

Abbreviations

     
  • APP

    amyloid precursor protein

  •  
  • APP-BP1

    APP-binding protein-1

  •  
  • β-TrcP

    β-transducin repeat-containing protein

  •  
  • MALT1

    mucosa-associated lymphoid tissue lymphoma translocation protein 1

  •  
  • CBM

    complex, CARMA1–Bcl-10–MALT1 complex

  •  
  • Cdc34

    cell division cycle 34

  •  
  • CSN

    COP9 signalosome

  •  
  • CYLD

    cylindromatosis protein

  •  
  • IκB

    inhibitory κB

  •  
  • IKK

    IκB kinase

  •  
  • IL-1β

    interleukin-1β

  •  
  • NF-κB

    nuclear factor κB

  •  
  • NEMO

    NF-κB essential modulator

  •  
  • RING

    really interesting new gene

  •  
  • RBX

    RING box

  •  
  • RIP

    receptor-interacting protein

  •  
  • SCF

    Skp1/cullin/F-box

  •  
  • SUMO

    small ubiquitin-related modifier

  •  
  • TAK1

    TGF (transforming growth factor)-β-activated kinase 1

  •  
  • TLR

    Toll-like receptor

  •  
  • TNF

    tumour necrosis factor

  •  
  • TNFR

    TNF receptor

  •  
  • TRAF

    TNFR-associated factor

  •  
  • UBA3

    ubiquitin-like modifier activating enzyme 3

We apologize that many relevant papers could not be cited here due to space constraints. This work was funded by the Ramón y Cajal Program, Ministerio de Educación y Ciencia grants BFU 2005-04091 and BFU2006-12991, the FIS (Fondo de Investigaciones Sanitarias) CIBERehd, the Department of Industry, Tourism and Trade of the Government of the Autonomous Community of the Basque Country (Etortek Research Programs 2005/2006) and from the Innovation Technology Department of the Bizkaia Country.

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RNF4 is a poly-SUMO specific E3 ubiquitin ligase required for arsenic-induced PML degradation
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