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PRMT5 dimethylates R30 of the p65 subunit to activate NF-κB - PubMed

  • ️Tue Jan 01 2013

PRMT5 dimethylates R30 of the p65 subunit to activate NF-κB

Han Wei et al. Proc Natl Acad Sci U S A. 2013.

Abstract

The ubiquitous inducible transcription factor NF-κB plays central roles in immune and inflammatory responses and in tumorigenesis. Complex posttranslational modifications of the p65 subunit (RelA) are a major aspect of the extremely flexible regulation of NF-κB activity. Although phosphorylation, acetylation, ubiquitination, and lysine methylation of NF-κB have been well described, arginine methylation has not yet been found. We now report that, in response to IL-1β, the p65 subunit of NF-κB is dimethylated on arginine 30 (R30) by protein-arginine methyltransferase 5 (PRMT5). Expression of the R30A and R30K mutants of p65 substantially decreased the ability of NF-κB to bind to κB elements and to drive gene expression. A model in which dimethyl R30 is placed into the crystal structure of p65 predicts new van der Waals contacts that stabilize intraprotein interactions and indirectly increase the affinity of p65 for DNA. PRMT5 was the only arginine methyltransferase that coprecipitated with p65, and its overexpression increased NF-κB activity, whereas PRMT5 knockdown had the opposite effect. 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 PRMT5 down. Many cytokine and chemokine genes are among these, and conditioned media from cells expressing the R30A mutant of p65 had much less NF-κB-inducing activity than media from cells expressing the wild-type protein. PRMT5 is overexpressed in many types of cancer, often to a striking degree, indicating that high levels of this enzyme may promote tumorigenesis, at least in part by facilitating NF-κB-induced gene expression.

Keywords: histone; mass spectrometry.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

R30 of p65 is dimethylated upon NF-κB activation, and this modification is essential for NF-κB function. (A) An analysis of tryptic peptides by MS shows that p65 is dimethylated on R30 upon NF-κB activation. A pure single band of p65 was digested in the gel and samples were analyzed by LC-MS/MS. A mass shift of +28 was observed for peptides 29–33. Tandem MS analysis further revealed that R30 on the N-terminal side of the peptide is dimethylated. (B) Western analysis of p65. (Left) Both WTp65-Flag and R30A-Flag were overexpressed at similar levels in 293IL1R cells. (Right) Both WTp65-Flag and R30K-Flag were overexpressed at similar levels in 293IL1R cells. (C) A coimmunoprecipitation experiment confirmed that R30 of p65 is dimethylated upon NF-κB activation. WTp65-Flag and R30A-Flag proteins were expressed in 293IL1R cells. Anti-Flag was used to pull the proteins down, and arginine methylation was detected by using anti-dimethyl arginine. (D, Left) EMSA was performed with extracts of 293IL1R cells, and of these cells in which WTp65 and R30A were overexpressed. The ability of NF-κB to bind to DNA was greatly enhanced upon over-expression of the WTp65 protein, but the R30A mutation severely decreased this ability. (Right) An EMSA assay showed that 293IL1R cells overexpressing the R30K mutant protein had less NF-κB binding activity than 293IL1R cells overexpressing WTp65. (E) Luciferase assay of NF-κB activity. 293IL1R cells were treated with 10 ng/mL of IL-1β for 4 h as a positive control. Expression of WTp65 significantly activated NF-κB activity in 293IL1R cells, but the R30A (Left) or R30K (Right) mutations greatly decreased this activity. The data represent the means ± SD from three independent experiments. *P < 0.01 vs. Control (Ctrl) group; †P < 0.01 vs. WT group.

Fig. 2.
Fig. 2.

PRMT5 binds to p65 and is responsible for NF-κB activation and dimethylation of R30. (A and B) Endogenous p65 coimmunoprecipitates with PRMT5. 293IL1R cells were treated with 10 ng/mL of IL-1β for 1 h or were untreated. PRMT5 binds to p65 only when NF-κB is activated. Reverse immunoprecipitation, using anti-PRMT5, gave a similar result. (C, Upper) Western assay showing either overexpression or shRNA-mediated knockdown of PRMT5 in 293IL1R cells. (Lower) Luciferase assay of NF-κB in 293IL1R cells treated with 10 ng/mL of IL-1β for different times, showing that reduced PRMT5 expression decreased and overexpression increased NF-κB activation. The data represent the means ± SD for three experiments. *P < 0.01 vs. Ctrl group. (D, Upper) Western assays, showing either overexpression (Upper Left) or shRNA-mediated knock down of PRMT5 (Upper Right) in colon cancer HT29 cells. (Lower) Luciferase assay of NF-κB in HT29 cells, showing that overexpression of PRMT5 further increased NF-κB activation, whereas reduced PRMT5 expression decreased NF-κB activation. The data represent the means ± SD for three experiments. *P < 0.01 vs. Ctrl group. (E) EMSA, showing that in response to IL-1β treatment, NF-κB DNA binding ability was significantly decreased in 293IL1R cells in which PRMT5 has been knocked down, compared with control cells (Left), whereas increased PRMT5 expression greatly enhanced NF-κB DNA binding ability (Right). (F) Coimmunoprecipitation and Western assays, showing that R30 of p65 is dimethylated by PRMT5 upon IL-1β treatment. Overexpression of PRMT5 increased the dimethylation of R30, but shRNA-mediated knock down had the opposite effect. (G) Luciferase assay of NF-κB activity, showing that the R30A mutation decreased NF-κB activation. Overexpression of PRMT5 activated NF-κB in control cells and in 293IL1R cells overexpressing WTp65, but not in these cells overexpressing the R30A mutant protein. The data represent the means ± SD from three experiments. *P < 0.01 vs. Ctrl group; †P < 0.01 vs. WTp65 group; #P < 0.01 vs. Ctrl, PRMT5 group; $P < 0.01 vs. WTp65, PRMT5 group.

Fig. 3.
Fig. 3.

Regulation of NF-κB–dependent gene expression by R30 methylation. (A) Illumina array data from 293IL1R cells overexpressing WTp65 or the R30A mutant protein. 293IL1R cells were used as the control. Among the 617 NF-κB–dependent genes induced by twofold or more in cells overexpressing WTp65 (WTp65 vs. 293IL1R), 464 (75%) were induced less well, by twofold or more, in cells with the R30A mutant protein (R30A/WT ≤ 0.5); the remaining 25% were not affected significantly. (B) A short list of typical NF-κB–inducible genes, showing that some were up-regulated by WTp65 by twofold or more (WT/Ctrl ≥ 2), but were up-regulated less well, by twofold or more, by the R30A mutant protein (R30A/WT ≤ 0.5). (C) Confirmation of Illumina array data by qPCR analysis. The expression of TNF-α, IL-8, and NFKBIA was tested, confirming that these genes were strongly induced upon IL-1β treatment or by the overexpression of WTp65 but not by the R30A mutant protein. The data represent the means ± SD from three independent experiments. *P < 0.01 vs. Ctrl group; †P < 0.01 vs. WT group. (D) An RT-PCR experiment further confirmed that the TNF-α, IL-8, and NFKBIA genes were induced in response to IL-1β in 293IL1R cells or by the overexpression of WTp65, but not by the overexpression of the R30A mutant protein.

Fig. 4.
Fig. 4.

Comparison of regulation of NF-κB dependent genes by PRMT5 and R30A. (A) Illumina array data, showing that 78% of genes were down-regulated by shPRMT5 by twofold or more, but the remaining 22% were not significantly affected. These percentages are similar to the percentages of R30A-dependent genes. (B) Illumina array data, suggesting that among the R30A dependent genes, about 85% were also PRMT5-dependent, meaning that they are also down-regulated by shPRMT5 by twofold or more, whereas the remaining 15% were not significantly affected. (C) A qPCR experiment was done to confirm that IL1A and TRAF1 were induced by the overexpression of WTp65, compared with control cells. However, this induction was greatly decreased after either R30A mutation or knockdown of PRMT5 by shRNA. Data represent the means ± SD for three independent experiments. *P < 0.01 vs. Ctrl group; †P < 0.01 vs. WT group.

Fig. 5.
Fig. 5.

Crystal structure of p65 showing that methylated R30 can mediate van der Waals contacts with both F184 and D277 to enhance its binding to DNA. (Right) Ribbon presentation of the RelA:κB DNA complex. A protein–protein interface away from the protein-DNA interface is highlighted to reveal the amino acid residues that are involved. Residues in red make DNA contacts. (Left) Surface presentation of the residues at the protein–protein interface. Dotted lines show that the atoms are in H-bonding distance. Methylated R30 can mediate van der Waals contacts with both F184 and D277.

Fig. 6.
Fig. 6.

A model. In addition to previously known regulatory pathways, NF-κB is regulated by PRMT5-mediated methylation of p65 on R30, which affects the expression of many NF-κB–induced genes.

Fig. 7.
Fig. 7.

PRMT5 is substantially overexpressed in cancers. (A) GeneNote data showing that PRMT5 is overexpressed in cancers of the thymus, bone marrow, blood, brain, kidney, lung, colon, bladder, liver, pancreas, prostate, skin, breast, salivary gland, ovary, and cervix, with the most striking overexpression in colon cancer. (B) Comparison of PRMT5 expression in 19 colon/colorectal vs. normal datasets in Oncomine. The intensity of the red signal indicates the level of overexpression.

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