Received Date: Janyary 13, 2014; Accepted Date: February 07, 2014; Published Date: February 12, 2014
Citation: Wei H, Mundade R, Lu T (2014) Regulation of NF-κB: Arginine Methylation Takes the Stage. J Mol Genet Med 8:098 doi:10.4172/1747-0862.1000098
Copyright: © 2014 Wei H, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Nuclear factor κB (NF-κB) is a family of transcription factors that play central roles in multiple biological functions, including immune and inflammatory responses and tumorigenesis. The regulation of the activity of NF-κB is very sophisticated. Post-translational modifications of NF-κB have long been known to play an essential role in the regulation of NF-κB activity. Since the p65 subunit (RelA) is the major subunit of the typical NF-κB heterodimer, more attention has been recently devoted to how p65 is regulated by the post-translational modifications. In this review, we provide an overview of the most recent developments in the regulation of p65 by post-translational modifications.
Arginine methylation; Epigenetic regulation; NF-κB; Post-translational modifications
Signaling through NF-κB plays an essential role in many disease conditions. Therefore, understanding elements that govern NF-κB has the potential to reveal targets for intervention to ameliorate a variety of diseases from cancer to inflammation. This review focuses on identifying new advances in our understanding of how NF-κB signaling is controlled.
The transcriptional factor NF-κB family includes p65 (RelA), RelB, c-Rel, p50/p105 (NF-κB1), and p52/p100 (NF-κB2). All proteins of the NF-κB family share a Rel homology domain (RHD) in their N-terminus, which is required for their dimerization, nuclear targeting, binding to DNA, and interaction with its inhibitors, the IκB proteins (IκBs) . A subgroup of NF-κB family members, namely the Rel proteins, including p65, RelB and c-Rel, also contain an additional carboxyterminal transactivation domain (TAD). The prototype of NF-κB is the heterodimer of p65 and p50 subunits. In unstimulated cells, NF-κB exists as homo- or heterodimers by binding to IκBs . There are seven IκB family members: IκBα, IκBβ, IκBγ, IκBε, BCL-3, and the precursor proteins p100 and p105, that bind to the RHD domains of NF-κB dimers . Each IκB interacts with their specific NF-κB dimers, covers their nuclear localization signals, and thus keeps NF-κB in an inactive state in cytoplasm. NF-κB has both canonical and non-canonical activation pathways . In the canonical NF-κB signaling pathway, extracellular signals such as proinflammatory cytokines activate IκB kinase (IKK), which phosphorylates IκBα, leading to its ubiquitinationand degradation by proteasomes . The released NF-κB (mostly p65/ p50 heterodimers) translocates into the nucleus, binds to DNA, and regulates the expression of multiple inflammatory and innate immune genes . In the non-canonical pathway, phosphorylation of p100 on its C-terminal helps the processing of p100 to p52 . The freed NF- κB (mostly p52/RelB heterodimers) translocates into the nucleus and regulates the expression of genes, many related to the development and maintenance of the immune system .
The NF-κB pathway is regulated by multiple post-translational modifications including ubiquitination, phosphorylation, acetylation, sumoylation, and nitrosylationetc . These regulatory modifications can vary with different NF-κB stimulators and even the same modifications may display quite different effects . In this review, we will only focus on the development of the post-translational modifications regarding p65 since p65 is the major subunit of NF-κB.
Phosphorylation of p65
Phosphorylation of p65 was nicely summarized by Perkins . Since then, only one more new serine (S) site, S547, has been identified . Here, we only name a few examples. For instance, three phosphorylated serine sites have been found in the RHD domain (amino acid 1-301) of p65, including S205, 276, and 281 [6-8]. Phosphorylation of S276 is catalyzed by either protein kinase K (PKA)  or mitogen- and stressactivated protein kinase 1 (MSK1)  in different cell systems, which in turn leads to the enhanced NF-κB transcriptional activity. Another study demonstrated that in response to lipopolysaccharide (LPS), S205 and 278 can be phosphorylated by unknown serine/threonine-directed protein kinases, which further increase NF-κB transcriptional activity . In addition to the RHD domain, four other serines were found to be phosphorylated in the TAD domain (amino acid 428-551): S468, 529, 536, and 547 [5,9-13]. Most phosphorylations on serine serve as an NF-κB activating signal. For example, upon TNFα treatment, S529 is phosphorylated by casein kinase II (CKII) which leads to the increased transactivation potential of NF-κB ; whereas S536 phosphorylation by the IKKβ increased p65 transcriptional activity in monocytes/ macrophages . S468 was found to be phosphorylated by IKKε , IKKβ , or GSK3β  in different cell systems. In response to T-cell co-stimulation, IKKε mediates inducible phosphorylation of S468, which enhance p65 transcriptional activity without influencing DNA binding ability . However, S468 can also inhibit p65 transactivation. For example, phosphorylation of S468 by GSK-3β negatively regulates NF-κB activity in unstimulated HeLa cells . Also phosphorylation of S468 catalyzed by IKKβ deceases NF-κB-dependent transcription in response to TNF-α and IL-1β treatment . By using p65S468Areconstituted p65−/− mouse embryonic fibroblasts (MEFs), Schwabe and Sakurai  found that S468A mutant significantly increases NF-κB activity after TNF-α and IL-1β treatment in comparison to wtp65-reconstituted MEFs . Other examples also suggest that phosphorylation on p65 is not always seen as an activation signal. A new study shows that phosphorylation of S547 by Ataxia telangiectasia mutated (ATM) represses NF-κB-dependent transcription in HEK-293 cells in response to DNA double-strand breaks (DSB) . Besides the 7 serine sites located on either the RHD or TAD domains described above, S311, a site located in the linker region (amino acid 302-427) between the RHD and TAD domains, is phosphorylated by the atypical protein kinase C ζ (PKCζ) . This important phosphorylation is required for CBP-enhanced NF-κB transcriptional activity in embryo fibroblasts (EFs) in response to TNFα stimulation .
In addition to serine phosphorylation, another major amino acid player in phosphorylation is threonine. To date, three threonines have been reported to be phosphorylated with different stimulations, including T254  in the RHD domain, and T435 , 505  in the TAD domain of p65. For instance, in response to cisplatin, T505 is phosphorylated by the checkpoint kinase Chk1  leading to the proapoptotic effect of p65 and negatively regulating autophagy, cellular proliferation, and migration .
Therefore, the above examples further illustrate the significant importance of phosphorylation in the regulation of the p65 subunit of NF-κB.
Acetylation of p65
As illustrated by Perkins , five acetylated lysine (K) sites have been identified on p65. They are K122, 123, 218, and 221, all located in the RHD domain, and K310, which is located in the linker region of p65. From 2006 to present, no additional acetylation site has been identified on p65. Chen et al. showed that in response to TNFα, K310 is acetylated by p300/CBP . This modification is required for the full transcriptional activity of p65 . In another report, K218, 221 are also seen to be acetylated by p300/CBP . Acetylation of K221 enhances p65 DNA binding, and in conjunction with acetylation of K218, blocks the interaction of p65 with newly synthesized IκBα, thereby, activating NF-κB. Acetylation at K122 and 123 by both p300 and p300/CBP-associated factor (PCAF) decreases the DNA binding ability, and helps the removal of NF-κB and subsequent export to the cytoplasm by IκBα .
In addition to the above modifications, it has also been seen that suppressor of cytokine signaling 1 (SOCS1) catalyzes the ubiquitination on amino acids 220–335 of p65 which results in the p65 degradation and negative regulation of NF-κB inducible genes expression . Nitration on tyrosine Y66 and Y152 of p65 triggered by nitric oxide treatment inhibits NF-κB activity . More details regarding other types of modifications of p65 can be found in the thorough review by Perkins .
As shown in Figure 1, beyond the more traditional modifications that are mentioned above, in the past few years, six methylated K sites have been identified on p65. They are: K37, 218, 221, 310, 314 and 315 [24-28]. In an attempt to identify the novel regulators of NF-κB by using a novel genetic approach, Lu et al. [25,29-31] discovered that the nuclear receptor binding SET domain protein 1 (NSD1) and the demethylase F-box leucine-rich protein 11 (FBXL11) regulate NF-κB through the reversible methylation of K218 and 221 of p65. Zhang et al.  reported that the plant homeodomain finger protein 20 (PHF20, also called glioma-expressed antigen 20 promotes NF-κB transcriptional activity by interacting with methylated p65 at K218 and 221. The interaction between PHF20 and methylated p65 blocks the recruitment of phosphatase PP2A to maintain the p65 phosphorylation status. Ea and Baltimore  reported that, upon activation of NF- κB by TNFα, Set9 monomethylates p65 at K37, and this epigenetic modification regulates the promoter binding of p65 to a subgroup of genes, such as IκBα, IP-10, and TNFα. The induction of IP-10 and TNFα was greatly reduced in p65−/−MEF cells that express K37Q mutant as compared to those that are reconstituted with wild type p65 . Yang et al. reported that p65 is monomethylated by Set9 at K314 and 315 , leading to the decreased NF-κB activity and its target gene expression. Levy et al. identified that SETD6 monomethylates p65 on K310, leading to the induction of a repressed state at NF-κB target genes through the binding of G9a-like protein in U2OS cells . Why does p65 need the methylation on multiple K sites? Recently, we studied the effect of methylation on K37 and K218/221 sites of p65 , and we found that mutation on K218/221 greatly reduced the expression of ~50% of NF-κB inducible genes, whereas the K37Q mutation reduced the expression of only ~25% of NF-κB inducible genes. Chromatin Immunoprecipitation sequencing (CHIP-Seq) analysis showed that the mutant of K218/221Q greatly reduces the affinity of p65 for many promoters, and that the K37Q mutation does not. Structural modeling shows that the newly introduced methyl groups of K218/221 interact directly with DNA to increase the affinity of p65 for specific κB sites. Thus, the K218/221 and K37 mutants have dramatically different effects because methylations of these residues affect different genes by distinct mechanisms . Furthermore, it is worth noting that it is a normal phenomenon to see that the same site of lysine can be either methylated or acetylated on the same protein, such as K218, 221 [20,25,32] and K310 [19,27] on p65. Besides NF-κB, there are numerous examples of the same amino acid being altered by different modifications. For instance, tumor suppressor p53 has been found to be methylated and acetylated on the same lysine sites including K370, 372 and 382, which leads to quite different biological functions . Furthermore, histone subunits, such as histone H2BK5, H3K9, 23, 27, 36 and H4K12 and 20 are all found to be modified by either methylation or acetylation on the same lysine with quite different biological consequences . This phenomenon further highlights the complexity of the posttranslational modifications of proteins.
Figure shows schematic diagram of the principal structural motifs of p65: Rel Homology domain (RHD, amino acid 19-301), Transactivation domain (TAD, amino acid 428-551) and the linker region (amino acid 302-427). The mapped sites are the post-translation modifications in p65; only phosphorylation, acetylation and methylation (Mono- or Di-methylation) sites are shown here.
Figure 1: Post-translational modifications of p65 .
Most recently, our lab discovered that the arginine (R) 30 of p65 (Figure 1) can be dimethylated by the protein arginine methyltransferase 5 (PRMT5), leading to activation of NF-κB . Microarray analysis revealed that ~85% of the NF-κB–inducible genes that are down regulated by the R30A mutation are similarly down regulated by knocking down PRMT5. This extremely novel finding suggests that, like tumor suppressor p53 , methylation of arginine of p65 also plays an essential role in the regulation of NF-κB. We predicted the crystal structure of p65 showing that methylated R30 can mediate Van der Waals contacts therefore increasing the ability of p65 to bind to DNA and consequently, affect gene expression . PRMT5 is a member of the PRMTs superfamily. Since PRMT5 is frequently seen overexpressed—often to a striking degree—in many types of cancer, such as colon, ovary, kidney, lung, bladder, liver, pancreas, breast, prostate, cervix, and skin, we suggest that high levels of this enzyme may promote tumorigenesis, at least in part by facilitating NF-κBinduced gene expression .
As we described above, post-translational modifications of NF-κB are complex and diverse. These modifications determine the activity of NF-κB and regulate its gene expression. Among all these known modifications, acetylation and methylation are the most attractive ones, as both are catalyzed by the histone modifying enzymes, a class of enzymes proven to be more promising druggable targets than others. Some inhibitors for histone deacetylase (HDAC) have already been developed and approved by US Food and Drug Administration (FDA), but with limited treatment outcome. Therefore, there is urgent need for identifying more promising druggable targets than HDAC. Since methylation is the newest type of modification found on NF-κB, this makes histone methyltransferases or demethylases the hottest drug targets today .
Based on the information regarding novel methylation sites (Figure 1) on NF-κB and their catalyzing enzymes, we predict that in different cell systems, histone methylasesNSD1, Set9 and PRMT5 could become promising druggable targets for small inhibitors that will decrease the activity of NF-κB (Figure 2). On the other hand, targeted activation of Set6D, FBXL11 or Set9 (in different cell system, ) by small molecules may negatively regulate NF-κB activity (Figure 2).
Figure 2: Predicted potential druggable targets through regulation of p65 methylation.We predict that NF-κB can be inhibited either by blocking the activities of the histone modifying enzymes, such as PRMT5, SET9 , NSD1, and SETD6, or by targeted activation of histone modifying enzymes FBXL11, or SETD6, or SET9 (reported in different cell system, [ 28]). Therefore, these enzymes could be potential druggable targets for controlling the NF-κB activity in diseases.
It is worth of noting that the identification of PRMT5 as the enzyme that can methylate R30 of p65 has been particularly important in shedding light on this previously under studied area due to the fact this is the first and only R residue that has been reported to be methylated on NF-κB. Like lysine methylation of p65, it would not be surprising if we were to see more methylated arginine sites of p65 subunit of NF-κB identified in the years to come. Tangled with other post-translational modifications of p65, known or to be discovered, as well as the modifications of other subunits of NF-κB, the fine-tuned regulation of NF-κB is a fertile field for the inspired researcher to explore.
We thank Ms. Lisa King at the Department of Pharmacology and Toxicology at Indiana University School of Medicine for her generous and professional help with revising this review. The research is supported by grants 23-862-07TL (to TL) and 036433730102 (to TL).
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