COMMD PROTEINS AND THE CONTROL OF THE NF-κB PATHWAY

Abstract

The COMMD family of proteins represents a recently discovered set of evolutionarily conserved factors characterized by the presence of a defining carboxy-terminal COMM domain. In vertebrates there are ten members of the family, and among their emerging functions, the control of the transcription factor NF-κB has been most extensively studied. NF-κB plays a critical role in a number of homeostatic processes in multicellular organisms, including the regulation of immunity and cell survival. COMMD proteins inhibit NF-κB mediated gene expression and studies on the mechanism of action of COMMD1 reveal that it controls the ubiquitination of NF-κB subunits, and event linked to transcriptional termination. COMMD1 binds to a multimeric ubiquitin ligase containing Elongins B/C, Cul2 and SOCS1 (ECS^SOCS1). In this complex, COMMD1 facilitates the binding of NF-κB to the ligase, thereby promoting their ubiquitination and degradation. Additional insights gained from these studies indicate that COMMD proteins likely play a broader role in cellular homeostasis through their participation in the ubiquitination pathway.

NF-κB: Current Paradigm

NF-κB is a dimeric transcription factor formed by members of a highly conserved family of proteins that share a ~ 300 amino acid sequence termed the Rel Homology Domain (RHD). In mammals, there are five genes that encode members of this family: RELA, RELB, REL, NFKB1 and NFKB2. Through transcriptional regulation of many gene products, NF-κB participates in a number of biologic processes including innate and adaptive immune responses, programmed cell death, transcriptional regulation of viral replication, cell cycle progression, and oncogenesis .^1–5

NF-κB mediated gene expression is regulated to a large extent by cytoplasmic sequestration of the NF-κB complex. In the canonical NF-κB pathway, this is the result of interactions between IκB proteins and NF-κB complexes (Figure 1).^{6, 7} Activation of a multimeric kinase known as the IκB Kinase (IKK) complex, results in phosphorylation of IκB. Once phosphorylated, IκB is ubiquitinated by a multimeric ubiquitin ligase containing Cul1 (known as SCF^βTrCp), targeting it for proteasomal degradation.^{8, 9} This enables the translocation of NF-κB complexes to the nucleus where they bind to cognate DNA sequences present in an array of promoters and can recruit the transcriptional machinery necessary for induction of gene expression. This is mediated through a series of complex events at the chromatin level that involve the removal of repressive complexes containing HDACs and their replacement with NF-κB in association with various transcriptional co-activators.^10–13 Once transcription has occurred, termination of NF-κB activity is also an important regulatory step. This is largely mediated by re-synthesis of IκB proteins, which facilitate nuclear export of NF-κB.¹⁴ In addition, ubiquitination of chromatin-bound NF-κB has been shown to be required for termination of NF-κB dependent gene expression, and the mechanism responsible for this ubiquitination event has begun to be elucidated.^{15, 16}

Figure 1. Schematic representation of the NF-κB pathway.

NF-κB exists as dimers in resting cells associated with the IκB proteins. Cell stimulation by a broad number of factors promotes the activation of the IKK complex. IκB proteins then become phosphorylated by IKK, resulting in their ubiqutination and proteasomal degradation. This enables NF-κB to translocate into the nucleus and induce expression of pro-inflammatory genes.

COMMD1 as an inhibitor of NF-κB

In search for regulators of the anti-apoptotic factor XIAP, we identified an interaction between XIAP and the MURR1 gene product (now referred to as COMMD1).¹⁷ At the time of the initial identification of this interaction, the MURR1 locus had no known function, but soon thereafter, MURR1 was shown to participate in copper metabolism, as mutations in canine MURR1 result in a copper overload disorder in Bedlington terriers.^{18, 19} An analysis of the functional consequence of the XIAP-COMMD1 interaction demonstrated that COMMD1 had no impact in the anti-apoptotic properties of XIAP, but rather it blocked XIAP-mediated NF-κB activation.^{20, 21} As an extension of those observations, it was appreciated that COMMD1 functions as a more global inhibitor of NF-κB activation mediated by a variety of stimuli including TNF, IL1-β, phorbol esters, and ectopic expression of IKK subunits. Interestingly, COMMD1 binds to the NF-κB complex, a fact that is likely linked to its global effect on NF-κB mediated transcription.

More recently, we have demonstrated that COMMD1 controls the expression of a number of endogenous NF-κB inducible gene products.²² TNF stimulation of cells deficient in COMMD1 resulted in greater accumulation of various endogenous κB-inducible transcripts, including ICAM1. Furthermore, conditioned media from COMMD1 deficient cells promoted enhanced chemotaxis of freshly isolated peripheral blood mononuclear cells across a membrane barrier and this increase in chemotaxis rate correlated with heightened secretion of NF-κB inducible chemokines such as CCL2.

In addition to these effects of COMMD1 on NF-κB mediated events, a role for this factor in controlling the HIV-1 life cycle has been previously demonstrated.²³ Expression levels of COMMD1 in freshly isolated naive CD4⁺ lymphocytes modulated the rate of HIV-1 replication. Cells overexpressing COMMD1 demonstrated blunted HIV-1 replication, while decreased expression of COMMD1 following RNA interference (RNAi) resulted in greater rates of replication. This finding is consistent with the known role of NF-κB in the life cycle of HIV-1, as well as with gene expression data in animal models of SIV disease progression. Specifically, macaques that experienced slow disease progression after SIV infection had the highest levels of COMMD1 expression before infection and during the 7 weeks of disease progression, compared to animals with typical or accelerated rates of disease progression. Altogether, this establishes a role for COMMD1 in the control of NF-κB mediated transcription, which in turn regulates events such as pro-inflammatory gene expression and the rate of HIV-1 replication, and possibly others that remain to be elucidated.

Discovery of the COMMD protein family

A biochemical screen for COMMD1 associated factors revealed the presence of three COMMD1-associated proteins, which notably had sequence homology to COMMD1. Additional efforts to search for COMMD1 homologous proteins in public databases identified a total of 10 factors in vertebrates, which were subsequently designated as COMMD proteins (COpper Metabolism MURR1 Domain containing).²⁰ The defining characteristic of all COMMDs is the presence of a ~70 amino acid region of high homology in their extreme carboxy-terminal end, designated as the COMM domain (Figure 2). This motif serves as an interface for COMMD proteins to interact with each other as well as other proteins, including NF-κB. With the exception of COMMD6, which consists primarily of a COMM domain, all other COMMD proteins possess additional amino-terminal structures which are not homologous amongst family members, but remain highly conserved across multiple species. The predicted structure of the amino-termini of all COMMDs suggests the presence of tandem α-helices. This is consistent with the recently reported solution structure of the amino-terminus of COMMD1.²⁴ However, the structure of the COMM domain itself remains to be elucidated.

Figure 2. Schematic representation of the COMMD family of proteins.

Shown in red is the conserved COMM domain along with the respective amino acid length of each protein in humans.

In addition to their shared COMM domain, and their ability to interact with each other through this domain, other COMMD proteins can also bind to NF-κB subunits and inhibit NF-κB activation.^{20, 25} Cells transfected to express COMMD proteins demonstrated suppression of κB-dependent expression of a reporter gene in response to TNF, and decreased expression of COMMD1, 4 and 6 after RNAi resulted in enhanced TNF-induced expression of the NF-κB responsive gene BIRC3/c-IAP2. Interestingly, the COMM domain seems to play a critical role in suppresion of NF-κB, as mutations in the conserved tryptophan and proline residues within the COMM domain of COMMD6 abrogated the inhibtory activity of the molecule.²⁶

COMMD proteins suppress NF-κB in a manner distinct from IκB proteins

Stimulation-dependent degradation of the IκB proteins represents a major pathway that allows translocation of NF-κB dimers to the nuclear compartment of cells, a critical step for the initiation of κB-mediated gene expression. Turnover of IκB proteins is mediated by a Cul1-containing multimeric ubiquitin ligase called SCF^β-TrCP, which promotes poly-ubiquitination of the phosphorylated form of IκB, targeting it for proteasomal degradation.^{6, 27} While COMMD1 can stabilize phosphorylated IκB-κ presumably through an interaction with Cul1, this event does not seem to be dominant since nuclear translocation of RelA is unaffected by COMMD1 expression.²⁰ Interestingly, an additional indication that COMMD proteins operate in a different manner than IκB is the fact that COMMD1 interacts with an amino-terminal motif present in all NF-κB subunits that is distinct from the motif that the IκB proteins associate with.

In addition to cytosolic sequestration of NF-κB, other mechanisms of transcriptional suppression involve control of the activity of chromatin bound NF-κB subunits through various pathways.^{12, 13, 15, 28–31} Evidence indicates that indeed COMMD1 regulates NF-κB after its nuclear entry. Increased expression of COMMD1 decreased the binding of RelA to chromatin and conversely, decreased endogenous expression of COMMD1 after RNAi intensified the association of RelA to the promoter site. In addition to these findings, COMMD1 was found to be recruited to the promoter itself in a stimulus-dependent manner. However, the mechanism responsible for the effects of COMMD1 on RelA-chromatin interactions remained elusive until recently.

COMMD1 inhibits NF-κB through a ubiquitination pathway

Ubiquitination and proteasomal degradation of DNA-bound RelA has been shown to control RelA-chromatin interactions independent of the IκB pathway, similar to the described effects of COMMD1 on RelA-chromatin binding.^{15, 16} Therefore, the possibility that ubiquitination of RelA might be a mechanism for the inhibitory effect of COMMD1 on κB-mediated transcription was recently examined.²² Indeed, the aggregate of the data supports the conclusion that COMMD1 promotes NF-κB ubiquitination as cells transfected to express COMMD1 accumulated greater amounts of ubiquitinated RelA. Conversely, depletion of endogenous COMMD1 by RNAi decreased the amount of ubiquinated RelA recovered.

Although COMMD1 can accelerate the ubiquitination of RelA, it remained to be determined whether this was a direct effect on ubiquitination or an indirect effect that facilitates the ubiquitination of RelA. Given that COMMD proteins have no known catalytic activity, it was examined if COMMD1 could interact with an endogenous ubiquitin ligase. Endogenous COMMD1 immunoprecipitates can catalyze the formation of polyubiquitin chains in an in vitro ubiquitination reaction when provided with the necessary co-factors (E1 and E2 enzymes, ubiquitin, and ATP), indicating that COMMD1 associates with a complex that has E3 ubiquitin ligase activity. The identity of this complex(es) would likely explain its ability to affect RelA ubiquitination.

COMMD1 promotes NF-κB ubiquitination through a distinct multimeric ubiquitin ligase

It has been demonstrated that a protein called SOCS1 promotes the ubiquitination and proteasomal degradation of RelA, although the context of this activity was unclear.¹⁵ SOCS1 is part of a larger family of proteins containing the conserved carboxy-terminal SOCS box domain. ³² Through their SOCS-box, these factors associate with Cullin-containing multimeric ubiquitin ligases. These complexes are referred to as ECS and contain Elongins B and C, Cullin 2 or 5, and a SOCS box containing protein.³³

Given that COMMD1 promotes RelA ubiquitination and its immunoprecipitates contain ubiquitin ligase activity, the notion that COMMD1 may associate with the ECS^SOCS1 complex was examined. Indeed, endogenous COMMD1 co-precipitated with endogenous SOCS1 and Cul2, with the later interaction being an inducible event critical to the assembly of the mature ECS^SOCS1 complex. In addition, COMMD1 was shown to interact with all core components of the ECS^SOCS1 complex in co-transfection experiments, including Elongin C, Cul2, SOCS1, and Rbx1. Furthermore, the data indicate that COMMD1 requires the ECS^SOCS1 complex to promote the ubiquitination of RelA. Suppression of Cul2 or SOCS1 expression after RNA interference abrogated the COMMD1-mediated accumulation of ubiquitinated RelA in cells. In addition, COMMD1 immunoprecipitates from cells expressing ECS^SOCS1 and not control transfected cells, efficiently promoted polyubiquitination of RelA in an in vitro ubiquitination reaction. These data establish that the association of COMMD1 with ECS^SOCS1 facilitates ubiquitination of NF-κB subunits. Additional studies, we also demonstrated that binding of COMMD1 to the ECS^SOCS1 complex optimally targets RelA for proteasomal degradation by facilitating RelA binding to the ligase.

Conclusion

The majority of the work on the regulation of NF-κB has focused on how this transcription factor becomes activated to promote induction of gene expression.^{5, 34} This includes pathways that regulate the translocation of NF-κB from the cytosol to the nucleus and the assembly of complexes on chromatin that promote transcription. Once NF-κB activation has taken place, the termination of transcription is an equally important regulatory step, which is partly mediated by re-synthesis of IκB proteins and export of nuclear NF-κB complexes. However, other mechanisms that are critical for the termination of the NF-κB-response are beginning to be uncovered.

It has been demonstrated that ubiquitination and proteasomal degradation of chromatin-bound RelA is required for normal transcriptional termination independent of IκB mediated nuclear export. Furthermore, in the study by Saccani et al, this ubiquitination event required RelA binding to DNA and seemed to be orchestrated locally at the site of the gene promoter, as proteasome subunits were shown to be recruited to the promoter itself.¹⁶ However, the identity of the ubiquitin ligase responsible for this was not elucidated at that time. Ryo and collaborators demonstrated that the SOCS1 protein can mediate the ubiqutiination of RelA, although in this study, the precise context of this event was not further delineated.¹⁵ Interestingly, we identified that COMMD1 acts as an accessory subunit to a SOCS1 containing complex (known as ECS^SOCS1), that targets RelA for ubiquitination. We observed that COMMD1 deficiency led to more persistent nuclear accumulation of RelA following TNF stimulation, while it did not affect the early entry of NF-κB into the nucleus, consistent with its primarily nuclear role in this pathway. Furthermore, our data indicate that at least the COMMD1 subunit of this complex is recuited to chromatin in a stimulus dependent fashion, and that levels of COMMD1 control the duration of RelA-chromatin association.

A model that would bridge the findings of Saccani, Ryo and our own work suggests that in response to NF-κB recruitement to promoter sites, a ubiquitin ligase containing COMMD1 is eventually recruited to ubiquitinate DNA-bound RelA (Figure 3). Importantly, this ligase also undergoes assembly in response to NF-κB activation, as evidenced by increased interaction between COMMD1 and Cul2, the main scaffold protein in the complex. This inducible interaction peaks at about 2 hours after NF-κB activation, suggesting that COMMD1-directed ubiquitination of NF-κB may be critical for late transcriptional termination, after IκB-mediated export has been completed. Indeed, our data suggests that for a number of genes, NF-κB mediated transcription continues to take place even after the total levels of nuclear RelA have returned to the pre-stimulation state, suggesting that active complexes are probably retained in the nucleus and that ubiquitination is likely an important mechanism for their removal.

Figure 3. Model of COMMD1-mediated negative regulation of NF-κB.

Activation of the NF-κB pathway promotes ubiquitination and degradation of IκB proteins by the SCF^βTrCP ubiquitin ligase, enabling translocation of NF-κB dimers to the nucleus where they bind cognate promoter elements to induce gene expression. COMMD proteins in complex with the ECS^SOCS1 ubiquitin ligase subsequently is recruited to these promoter sites, facilitating poly-ubiquitination of NF-κB subunits. This event ultimately targets NF-κB for proteasomal degradation and termination of transcription.

The COMMD1-ECS^SOCS1 complex represents the second Cullin-containing ubiquitin ligase that regulates the NF-κB pathway. The Cul1-containing SCF^β-TrCP complex plays a critical role in the ubiquitination of IκB proteins and the precursors p100 and p105.³⁵ Interestingly, COMMD1 binds to additional Cullins, including Cul1, Cul2, Cul3 and Cul5, a fact that is not entirely surprising since the Cul2-COMMD1 interaction was mapped to the conserved Cullin-homology domain. Given the multitude of targets that these ubquitin ligases have, it can be speculated that COMMD1 likely plays a role outside of regulating NF-κB, and this may underly other reported activities of this protein such as its involvement in copper metabolism or the regulation of ENaC.^{17, 36} Similarly, the COMMD family contains 9 additional members which can interact with each other, suggesting the possibility of broader biologic roles for this protein family through their involvement in the ubiquitination cascade.