Role of Novel Serine 316 Phosphorylation of the p65 Subunit of NF-κB in Differential Gene Regulation

Abstract

Nuclear factor κB (NF-κB) is a central coordinator in immune and inflammatory responses. Constitutive NF-κB is often found in some types of cancers, contributing to oncogenesis and tumor progression. Therefore, knowing how NF-κB is regulated is important for its therapeutic control. Post-translational modification of the p65 subunit of NF-κB is a well known approach for its regulation. Here, we reported that in response to interleukin 1β, the p65 subunit of NF-κB is phosphorylated on the novel serine 316. Overexpression of S316A (serine 316 → alanine) mutant exhibited significantly reduced ability to activate NF-κB and decreased cell growth as compared with wtp65 (wild type p65). Moreover, conditioned media from cells expressing the S316A-p65 mutant had a considerably lower ability to induce NF-κB than that of wtp65. Our data suggested that phosphorylation of p65 on Ser-316 controls the activity and function of NF-κB. Importantly, we found that phosphorylation at the novel Ser-316 site and other two known phosphorylation sites, Ser-529 and Ser-536, either individually or cooperatively, regulated distinct groups of NF-κB-dependent genes, suggesting the unique role of each individual phosphorylation site on NF-κB-dependent gene regulation. Our novel findings provide an important piece of evidence regarding differential regulation of NF-κB-dependent genes through phosphorylation of different p65 serine residues, thus shedding light on novel mechanisms for the pathway-specific control of NF-κB. This knowledge is key to develop strategies for prevention and treatment of constitutive NF-κB-driven inflammatory diseases and cancers.

Keywords: NF-κB (NF-κB); mass spectrometry (MS); phosphorylation; post-translational modification (PTM); serine.

FIGURE 1.

Mass spectrometry identified Ser-316 phosphorylation of p65 in response to IL-1β. Tandem mass spectrometry (ms2) of precursor ions in the phosphorylated p65 peptide (amino acids 316–329; sequence SPFSGPTDPRPPPR, where S (red) indicates phosphorylated serine). Black lines indicate peptide cleavage. Compared with unmodified 316–329 peptide, an 80-Da mass shift was observed on precursor ion and b series ions such as b3, b7, and b8, but not on y series ions from y4 to y13, indicating phosphorylation of Ser-316.

FIGURE 2.

Effects of the S316A mutation on NF-κB activity. A, Western assays showing the overexpression of wtp65 and S316A-p65 in 293C6 cells (left panel) or in MEFp65^−/− cells (right panel). The molecular marker is marked on the left. Units are in kDa. B, luciferase assay of NF-κB activity. Overexpression of wtp65 significantly activated NF-κB, whereas overexpression of the S316A-p65 mutant protein reduced this activity in both 293C6 cells (left panel) and MEFp65^−/− cells (right panel). Results of triplicate luciferase assays are shown as the mean ± S.D. *, p < 0.01 versus control (Ctrl) group; #, p < 0.01 versus wt group. C, immunofluorescence, showing the localization of wtp65 and S316A-p65. Both FLAG-tagged wtp65 and S316A-p65 were probed with anti-FLAG antibody. DAPI was used for nuclear staining. In the wtp65 overexpression cell line, without IL-1β treatment the localization of p65 was observed in both cytoplasm and nucleus but mainly in the cytoplasm; however, after IL-1β treatment, p65 was largely translocated into the nucleus. In contrast, p65 translocation was substantially decreased in cell lines with S316A mutation in the presence or absence of IL-1β treatment, indicating that mutation at Ser-316 compromised the nuclear translocation of p65 subunit. Control 293C6 cell lines without overexpression of FLAG-tagged p65 did not show the expression of FLAG-tagged p65 and was used as a negative control.

FIGURE 3.

Regulation of NF-κB-dependent gene expression by Ser-316 phosphorylation. A, a short list of NF-κB-dependent genes that are down-regulated by S316A-p65 mutant. These genes encode proteins that have a broad range of functions. B, quantitative PCR analysis showing that TTLL2, USP28, SLC32A1, NKG7, and TRIM73 were induced by the overexpression of wtp65 as compared with control cells. However, this induction was greatly decreased after S316A-p65 mutation. The data represent the mean ± S.D. from three independent experiments. *, p < 0.01 versus control (Ctrl) group; #, p < 0.01 versus wt group.

FIGURE 4.

Important biological functions of Ser-316. A, assays of conditioned media showing that conditioned media from 293C6 cells (left panel) overexpressing wtp65 had much higher NF-κB-inducing activity than media from cells overexpressing the S316A-p65 mutant protein. Similar phenomenon was observed with MEFp65^−/− cells (right panel). Stable 293-NF-κB reporter cells were used. The data were normalized to the total number of cells that generated the conditioned media and to the total amounts of protein. The results of triplicate luciferase assays are shown as the mean ± S.D. *, p < 0.05 versus control (Ctrl) group; #, p < 0.05 versus wt group. B, analysis of cytokine expression in the conditioned media from 293C6 control, wtp65, or S316A-p65 by using human cytokine ELISA plate array. Total 32 cytokines/chemokines were tested. C, cell growth rate assay. Top panel, Western assays showing overexpression of wtp65 and Ser-316-p65 in 293C6 stable cells. The molecular marker is marked on the left. Units, kDa. Bottom panels: cell growth rate, showing that wtp65-overexpressing cells grew much more rapidly than either control or S316A-p65-overexpressing cells. The results of triplicate luciferase assays are shown as the mean ± S.D. *, p < 0.05 versus control group.

FIGURE 5.

Dynamic phosphorylation of Ser-529 and Ser-536 of p65 in response to IL-1β in 293C6 cells. A Western assay shows that upon IL-1β treatment, both Ser(P)-529 and Ser(P)-536-p65 were induced within 15 min then started to decay after 2 h. Meanwhile, IκB was degraded, indicating the activation of NF-κB. The molecular marker is marked on the left. Units, kDa.

FIGURE 6.

Differential regulation of NF-κB-dependent genes by phosphorylation of Ser-316, -529, and -536. A–C, microarray data show the different gene regulation effects by different p65 serine to alanine mutants, including S316A, S529A and S536A single mutants and S316/529, S316/536, S529/536 double mutants as well as the S316A/S529A/S536A triple mutant. In this experiment, the data showing the ratio of gene expression levels from mutant(s) p65 versus wtp65, the levels of gene expression from wtp65 sample were considered as 1. Overall, each single mutant was able to decrease 70∼80% that of NF-κB target gene expression. Double and triple mutants had slightly more dramatic effect than each single site mutant. D and E, phosphorylation of Ser-316, Ser-529, and Ser-536 can either commonly (all three sites) regulate some of NF-κB target genes or solely regulate their specific pools of genes. Moreover, two different sites, i.e. Ser-316/529, or Ser-316/536 or Ser-529/536 can commonly regulate some genes, which are not regulated by the third S site, suggesting a hieratical regulation pattern.

FIGURE 7.

CKI Phosphorylates p65 at Ser-316. A, HPRD website predicted CKI motif in position 316–319 of p65. B, NF-κB activity was determined by luciferase assay, showing that CKI inhibitor D4476 significantly decreased the NF-κB-inducing activity in both 293C6 cells treated with IL-1β and wtp65 overexpressing cells but had no notable effect on the S316A-p65 overexpressing cells, suggesting that D4476 functioned through Ser-316 phosphorylation. The data represent the mean ± S.D. from three independent experiments. *, p < 0.01 versus control +IL-1β (Ctrl+IL-1β) group; #, p < 0.01 versus wtp65 group.

FIGURE 8.

Hypothetical model. We hypothesize that upon treatment with IL-1β, Ser-316, Ser-529, or Ser-536 of p65 are phosphorylated by the same or different kinases, including CKI (inset: magnified image of the precise phosphorylation sites and their location in the p65 structure). Each phosphorylated site regulates the common core (∼80% of genes in our case) of NF-κB dependent genes. Additionally, each site further regulates ∼20% unique subgroups of genes (inset: illustrating common core genes and different specific subgroups of genes that are activated by each of the three phosphorylated sites individually or cooperatively). This regulation pattern not only will allow NF-κB to exhibit its major fundamental biological functions but also further provide NF-κB with more specificity and flexibility, thus leading to diversified NF-κB-driven biological consequences in specific cell systems under specific microenvironments and in response to a variety of different specific stimuli. TAD, transactivation domain.