Deleted in Breast Cancer 1 Suppresses B Cell Activation through RelB and Is Regulated by IKKα Phosphorylation

Sinyi Kong, Hongxin Dong, Jianxun Song, Muthusamy Thiruppathi, Bellur S. Prabhakar, Quan Qiu, Zhenghong Lin, Eduardo Chini, Bin Zhang and Deyu Fang
J Immunol October 15, 2015, 195 (8) 3685-3693; DOI: https://doi.org/10.4049/jimmunol.1500713

Abstract

Alternative NF-κB signaling is crucial for B cell activation and Ig production, and it is mainly regulated by the inhibitor of κ B kinase (IKK) regulatory complex. Dysregulation of alternative NF-κB signaling in B cells could therefore lead to hyperactive B cells and Ig overproduction. In our previous, study we found that deleted in breast cancer 1 (DBC1) is a suppressor of the alternative NF-κB pathway to attenuate B cell activation. In this study, we report that loss of DBC1 results in spontaneous overproduction of Ig in mice after 10 mo of age. Using a double mutant genetic model, we confirm that DBC1 suppresses B cell activation through RelB inhibition. At the molecular level, we show that DBC1 interacts with alternative NF-κB members RelB and p52 through its leucine zipper domain. In addition, phosphorylation of DBC1 at its C terminus by IKKα facilitates its interaction with RelB and IKKα, indicating that DBC1-mediated suppression of alternative NF-κB is regulated by IKKα. Our results define the molecular mechanism of DBC1 inhibition of alternative NF-κB activation in suppressing B cell activation.

Introduction

The NF-κB pathway is critical in many processes, such as cell survival, inflammatory cytokine signaling, and apoptosis (1–3). Although the canonical signaling pathway is highly expressed in most cell types, the alternative NF-κB pathway is central to a much smaller subset of cell types—namely bone, dendritic cells, and B cells (1, 4–6). In addition, the canonical and alternative NF-κB pathways have distinct functions (3). In B cells, the alternative NF-κB pathway regulates development from transitional to mature B cells, cell cycle entry, isotype switching and differentiation, and plasma cell survival (5, 7–13). As a result, either reduced or increased alternative NF-κB activation is associated with defective B cell response or B cell–mediated autoimmune diseases, respectively (5, 12, 14–17).

One of the main regulatory mechanisms of the NF-κB pathway is through the inhibitor of κ B kinase (IKK) regulatory complex, which consists of at least IKKα, IKKβ, or IKKγ (NF-κB essential modulator) (18). IKKα and IKKβ are both ∼750 aa in size, whereas IKKγ is 300 aa smaller in size, and has a distinct structure from the former two (18, 19). Although IKKα and IKKβ are structurally similar, knockout (KO) studies show that IKKα and IKKβ differentially regulate the alternative and canonical NF-κB pathways, respectively (18–21). IKKβ preferentially binds to and phosphorylates IĸBα, which is then degraded to release RelA: p50 heterodimers into the nucleus (18). However, IKKα phosphorylates the regulatory domain of p100, a necessary step to yield transcriptionally active RelB:p52 dimers (18, 21).

Deleted in breast cancer 1 (DBC1) was found to interact with IKKβ through a mass spectrometry screen (22). Although DBC1 was initially identified as a tumor suppressor, its role is cell-type dependent, as upregulated DBC1 levels are found in various other cancers (23–26). In addition to IKKβ, DBC1 has been shown to interact with various nuclear proteins, such as ER, AR, BRCA1, HDAC3, SUV39H1, and Sirt1, thus implicating its role in regulating transcription and epigenetic modification (27–33). We previously identified a novel role of DBC1 as a suppressor of B cell activation (34). In addition, through a microarray screen, we showed that in DBC1 KO B cells, RelB activity was significantly upregulated (34). In this study, we report that the loss of DBC1 in mice leads to spontaneous dysregulation of B cells at 10 mo of age, leading to increased production of autoreactive Ig. Furthermore, we confirm that DBC1 suppresses B cell regulation through RelB using a double mutant genetic mouse model. At the molecular level, we show that DBC1 interacts with both alternative NF-κB members RelB and p52, as well as its regulator IKKα. Lastly, although the N-terminus leucine zipper (LZ) domain of DBC1 is required for its interaction with RelB, phosphorylation of DBC1 at its C terminus by IKKα is required for its interaction with both RelB and IKKα. Our study further defines the molecular mechanism of DBC1 suppression of NF-κB and its role in B cell regulation.

Materials and Methods

Mouse and cell lines

HEK293T and NIH3T3 cells lines were maintained in DMEM (Life Technologies) supplemented with 10% FCS and 1% penicillin/streptomycin. EL4 cell line was maintained in RPMI 1640 supplemented with 10% FCS and 1% penicillin/streptomycin. Dbc1−/− mice were gifted by the Chini laboratory, and further backcrossed to C57BL/6 background for more than five generations. Relbshep/shep mice were purchased from Jackson Laboratories and crossed with DBC1 KO mice to generate Dbc1−/− Relbshep/shep double mutant mice with mixed genetic background. For all experiments, littermates were used as controls. All mice used in this study were maintained and used at the Northwestern University mouse facility under pathogen-free conditions according to institutional guidelines and animal study proposals approved by the Institutional Animal Care and Use Committee.

Plasmids, Abs, and reagents

PcDNA-Myc-DBC1 plasmid was purchased from Addgene. Truncation mutants were subcloned into pCMV-Myc (Clontech), and Myc-DBC1-SA mutant was generated by site-directed mutagenesis with the Advantage GC Rich PCR kit (Clontech) using standard protocol. RelA-cFlag pcDNA3, RelB-cFlag pcDNA3, C-Rel RHD-cFlag-pcDNA3, p50-cFlag-pcDNA3, p52-cFlag pcDNA3, pCR-Flag-IKKα, and pCR-Flag-IKKβ plasmids were purchased from Addgene. Abs used for immunoblotting and coimmunoprecipitation were anti-p30/DBC1 (Bethyl Laboratories), anti-RelB(C-19) (D-4), p100/p52 (C-5), Myc (A-14), (9E10), IKKα/β (H-470; Santa Cruz), anti-Flag (F7425; F1804; Sigma), anti-Tubulin (DM1A; Calbiochem) anti-phosphoserine (AB1603), and phosphothreonine (AB1607; Millipore).

Primary B cell isolation and culture

Primary B cells were negatively isolated from 8–12-wk-old mice using Dynabeads Mouse CD43 (Untouched B cells; Life Technologies) per the manufacturer’s instructions. Primary B cells were maintained at 106/ml in RPMI 1640 (Dibco) supplemented with 10% FBS, 50 μM β-mercaptoethanol, 100 mM sodium pyruvate, 100 mM HEPES buffer, and 1% penicillin/streptomycin. B cells were activated with goat f(ab)2 anti-mouse IgM (10 μg/ml; Jackson ImmunoResearch), anti-CD40 (1 μg/ml; eBioscience) supplemented with IL4 (10 ng/ml), LPS (500 ng/ml), BAFF (100 ng/ml; Peprotech) as indicated.

CFSE proliferation assay

For cell proliferation and Ig production assays, purified B cells were stained with Cell Trace CFSE (5 μm; Life Technologies), and cultured at 106 cells/ml for 5 d with indicated stimuli. After 5 d, cells were subjected to flow cytometry and analysis.

Flow cytometry

Single-cell suspensions were Fc-blocked with anti-CD16/32 Ab (eBioscience), stained with the appropriate fluorophore-conjugated Abs, and collected by an Accuri C6 Flow Cytometer or FACSCanto (BD Biosciences). Fluorescence-labeled Abs used include FITC-conjugated anti-mouse IgA, PE-conjugated anti-CD138, allophycocyanin-conjugated anti-IgG1 (BD Biosciences), peridinin-chlorophyll Cy5.5-conjugated anti-B220 (BioLegend). For intracellular staining of Ig, cells were fixed and permeabilized using the CytoFix/Perm Kit (BD Biosciences) per the manufacturer’s instructions, incubated with 1:400× dilution of isolated serum in 1% BSA, and stained with the appropriate fluorophore-conjugated Abs.

Immunofluorescence staining

NIH3T3 cells were seeded on Poly-L-Lysine coated cover slips in six-well plates. Cells were fixed with 4% formaldehyde, permeabilized with 0.1% Triton-X100, and incubated with 1:400× diluted serum in 1% BSA for 1 h at room temperature (RT). Cells were washed and incubated with Alexa Fluor 594-conjugated anti mouse IgG (Invitrogen) and biotin-labeled anti-mouse IgA for 1 h at RT, followed by Alexafluor-488 avidin (Invitrogen) for 1 h at RT. Cells were stained with DAPI before visualization with the Nikon eclipse Ti Fluorescence microscope at original magnification ×40 (2-s exposure).

ELISA

Ninety-six–well flat-bottom plates were coated overnight at 4°C with 50 ng purified anti-mouse Abs (BioLegend) for the specific isotypes. Wells were then blocked with 1% BSA at RT for 2 h and then incubated with mouse sera at 1:200 to 1:10,000 dilutions overnight at 4°C. Wells were then incubated with biotin-coated secondary Abs against specific Ig for 1 h at RT, followed by avidin-HRP, and TMB substrate (Thermo Scientific). Absorbance at 450 nm was detected using a FilterMax F5 microplate reader (Molecular Devices).

Real-time PCR

Cells (106) were lysed in Trizol (Invitrogen), and RNA was isolated per the manufacturer’s instructions. Isolated RNA (1 μg) was then reversed transcribed using qScript cDNA Synthesis Kit (Quanta BioSciences). Real-time PCR was performed in duplicate wells using the iCycler Sequence and SsoFast SYBR Green Supermix (BioRad).

Statistical analysis

Student t test was used to calculate statistical significance for ELISA, flow cytometry, and Western blot densitometry analysis. For CFSE, ELISA and quantitative PCR analysis of DKO (RelB/DBC1 double KO) mice, the Tukey-Kramer method was used for calculating statistical significance. A p value ≤0.05 was considered significant.

Results

Loss of DBC1 leads to increased Ig production in 10-mo-old mice

In our previous study, we showed that loss of DBC1 in mice leads to increased autoantibody production when induced with experimental autoimmune myasthenia gravis (34). Through our study, we proposed that the tight regulation of B cell activation is lost in DBC1 KO mice. Interestingly, we now find that at 10 mo of age, DBC1 KO mice spontaneously produce increased levels of autoreactive Ig. Using an indirect immunofluorescence staining method, serum from 10-mo-old DBC1 KO mice revealed higher levels of autoreactive IgG and IgA toward the murine fibroblast cell line NIH 3T3 (Fig. 1A, 1B). Specifically, autoreactive IgG was undetectable from wild type (WT) serum at 1:800 dilution. In contrast, serum from DBC1 KO mice had persistently high levels of autoreactive IgG across all dilutions (Fig. 1A, 1B, top panels). Likewise, for the IgA isotype, WT serum had barely detectable levels of fibroblast-bound IgA at 1:400 dilution, whereas autoreactive IgA from DBC1 KO mice remained at high levels, even at 1:800 dilution (Fig. 1A, bottom panel; Fig. 1B, right graph).

FIGURE 1.
FIGURE 1.

DBC1 KO mice spontaneously increase production of serum Ig at 10 mo. (A) Autoreactive IgG (red) and IgA (green) from the sera of 10-mo-old WT and DBC1 KO mice was detected by indirect IF staining using the NIH 3T3 cell line. Original magnification ×40. (B) Mean fluorescence density from (A). (C) Serum from WT and DBC1 KO mice were tested for IgG1 and IgA reactivity toward surface (top) and intracellular (bottom) autoantigens in EL4 murine T lymphoma cell line, and detected by flow cytometry. (D) Mean fluorescence intensity (MFI) of EL4-bound Ig as detected in (C). (E) Blood serum was isolated from 10-mo-old WT and DBC1 KO mice, and serum Ig levels for the indicated isotypes were measured by ELISA. (F) Autoreactive IgG and IgA as measured in (A) normalized to total IgG and IgA levels (fluorescence density [arbitrary unit]/μg). n = 5, error bars represent SEM. *p < 0.05, ***p < 0.001.

We confirmed the increase of autoreactive Ig in DBC1 KO mouse serum by testing reactivity toward the murine T cell lymphoma cell line EL4. Similarly, significantly higher levels of autoreactive IgG and IgA levels in DBC1 KO serum were detected when analyzed with flow cytometry. Furthermore, DBC1 KO Ig could bind to both surface and intracellular autoantigens, as shown by flow analysis of differential surface staining and intracellular staining of EL4 cell line (Fig. 1C, 1D). The increased levels of autoreactive Ig correlated with significantly higher levels of total serum Ig, specifically IgG1, IgA, and IgE isotypes (Fig. 1E). However, because of increased total Ig levels in DBC1 KO mice, DBC1 KO mice autoantibody levels normalized to total Ab levels (fluorescence relative density [arbitrary unit]/Total Ab concentration) are not significantly different from WT littermates (Fig. 1F). Taken together, the data indicate that the loss of DBC1 in mice leads to a loss of tight regulation of B cell activation, and spontaneous increased production of autoreactive and total Ig at 10 mo of age.

DBC1 interacts with RelB and P52

Our findings prompted us to determine the molecular basis for the dysregulation of B cell activation in DBC1 KO mice. In our previous study, microarray data and chromatin immunoprecipitation (ChIP) experiments suggest that RelB activity is increased in DBC1 KO B cells (34). We also tested whether DBC1 suppresses RelB activity through binding with RelB. We cotransfected Myc-tagged DBC1 with each of the Flag-tagged NF-κB members into HEK293T cells, and tested for interaction by coimmunoprecipitation (Co-IP). We observed that DBC1 underwent Co-IP with RelB and p52 (Fig. 2A, 2B). In contrast, we did not detect an interaction between DBC1 and RelA, c-Rel, or p50 (Fig. 2A, 2B). In addition, the interaction of DBC1 with RelB and p52 was confirmed in mouse primary splenic B cells by immunoprecipitation of DBC1 (Fig. 2C, 2D). RelB interacted with DBC1 in primary splenic B cells, and this interaction was reduced upon 16 h of CD40 stimulation (Fig. 2C, 2D). Similarly, we detected an interaction between DBC1 and p52 in naive primary B cells (Fig. 2C, second panel; Fig. 2D). Interestingly, upon CD40 activation, DBC1 partially reduces its interaction with p52, and increases its interaction with the inactive precursor p100 instead (Fig. 2C, second panel; Fig. 2D). These results thus indicate that DBC1 interacts with the alternative NF-κB members RelB and p52, supporting our hypothesis that DBC1 regulates B cell function by selectively modulating the transcriptional activity of the alternative NF-κB pathway.

FIGURE 2.
FIGURE 2.

DBC1 interacts with RelB and p52. (A) Flag-tagged RelA, RelB, c-Rel, p50, and p52 were each cotransfected with myc-tagged DBC1 into HEK293T cells. Cell lysates were then immunoprecipitated using anti-Flag Ab. Interaction with DBC1 was detected by immunoblotting with anti-Myc Ab. (B) Densitometry analysis of (A) based on five independent trials. (C) Splenic B cells from WT mice were activated with anti-CD40 for 0, 1, and 16 h. Next, cell lysates were immunoprecipitated with anti-DBC1 Ab. Interactions between endogenous DBC1 and RelB and p52 were detected by immunoblotting. Normal rabbit IgG serves as negative control. (D) Densitometry analysis of (C) based on six independent trials. *p < 0.05.

The LZ domain of DBC1 is necessary and sufficient for interaction with RelB

To characterize the molecular mechanism of DBC1 suppression of RelB, we mapped the interaction domains of DBC1 and RelB. DBC1 protein has a LZ, putative hydrolase domain, inactive EF hand, Nuclear Localization Sequence and coiled-coil domain (Fig. 3A) (31, 34). We generated five truncated mutants of DBC1 (Fig. 3A) and then cotransfected Flag-tagged RelB with either Myc-tagged full-length DBC1 or each of the five Myc-tagged truncated mutants of DBC1 into HEK293T cells (Fig. 3B). Co-IP results show that the LZ domain of DBC1 is required and sufficient for interaction with RelB (Fig. 3B). Specifically, deletion of the N terminus led to a loss of DBC1 interaction with RelB, whereas expression of the N terminus of DBC1 alone was sufficient for interaction with RelB (Fig. 3B, lanes 3 and 4). Interestingly, we observed that when the center hydrolase domain was expressed along with the LZ domain, interaction between DBC1 and RelB was significantly reduced compared with interaction with the LZ domain of DBC1 alone, suggesting that a regulatory domain is present at the C terminus to the LZ domain of DBC1 (Fig. 3B, 3C).

FIGURE 3.
FIGURE 3.

The LZ of DBC1 is necessary and sufficient for interaction with RelB. (A) Schematic of structure of full-length DBC1 and generated truncated mutants. (B) Myc-tagged DBC1 and its truncated mutants were cotransfected with Flag-tagged RelB into HEK293T cells, and cell lysates were immunoprecipitated with anti-Myc Ab. Interaction with RelB was detected by Western blot using anti-Flag Ab (top panel). Whole cell lysate (WCL; bottom panel) served as loading control. (C) Densitometry analysis of (B). RelB coimmunoprecipitated with full-length DBC1—lane 2 of (B)—was used to normalize RelB coimmunoprecipitate levels with truncated mutants. n = 3; error bars represent SEM. *p < 0.05. CC, coiled-coil; EF, inactive EF hand; Hydrolase, putative hydrolase domain; NLS, nuclear localization signal.

Increased proliferation and Ig production of DBC1 KO mice is abrogated by deletion of RelB

To confirm that hyperactivation of DBC1 KO B cells is due to loss of DBC1 suppression of RelB in B cells, we crossbred DBC1 KO mice with RelBshep/shep (RelB μ) mice, which bear a spontaneous loss-of-function point mutation of RelB (35). Although RelB protein level was unaffected in the mutant (Fig. 4A), RelB function was significantly diminished in B cells from RelB shep/shep mice, because we could not detect mutant RelB DNA-binding activity using ChIP assay (Fig. 4B). As a result, at 6–8 wk of age, RelBshep/shep mice exhibit a similar but milder phenotype than RelB-null mice do in the hematopoietic compartment (12), specifically a slight reduction of mature B cell compartment in the bone marrow (Fig. 4C) and spleen (Fig. 4D), and an increase in B220−CD3ɛ−Mac1+ cells (Fig. 4D, 4E).

FIGURE 4.
FIGURE 4.

RelBshep/shep mutation leads to loss-of-function RelB protein. (A) Western blot analysis of RelB protein levels from two pairs of RelB+/shep and RelBshep/shep mice. GAPDH levels served as loading control. (B) B cells from RelB+/shep (WT) and RelBshep/shep (RelB μ) mice were activated with anti-CD40 overnight and subjected to ChIP analysis. RelB-bound target gene promoters were quantified by quantitative PCR. (C) Bone marrow cells were isolated from WT and RelBshep/shep mice and B220LO IgM−, B220LO IgM+, and B220HI IgM+ cells analyzed by flow cytometry (top panel). Mean percentages of B cell subpopulations of WT and RelBshep/shep mice are shown (bar graph in bottom panel) (D) Splenocytes were isolated WT and RelBshep/shep mice and analyzed for B220+, CD3ɛ+ and B220−CD3ɛ− Mac1+ cells. (E) Mean percentages of B cells, T cells and macrophages/monocytes as analyzed in (D). For (A) and (B), n = 5 from two independent experiments. For (C)–(E), n = 5. Error bars indicate SEM. *p < 0.05, **p < 0.01.

Naive B cells were then isolated from WT, Dbc1−/− (DBC1 KO), RelBshep/shep (RelB μ), and Dbc1−/−RelBshep/shep (DKO) mice and analyzed for proliferation and Ig production. Consistent with our hypothesis, when assayed for proliferation by CFSE, DBC1 KO B cells proliferated at a faster rate compared with WT when stimulated with RelB agonists anti-CD40 or BAFF (Fig. 5A, 5B). In contrast, RelB μ B cells had reduced proliferation compared with WT, as expected. Importantly, proliferation of Dbc1−/− RelBshep/shep (DKO) B cells were identical to RelB μ B cells, indicating that the loss of RelB function abrogates the hyperproliferative phenotype observed in DBC1 KO B cells (Fig. 5A–C). Consistent with the proliferation data, ELISA using culture supernatants reveal that IgG1 and IgA production was increased in DBC1 KO B cells, but not in RelB μ and DKO B cells (Fig. 5D). In addition, we previously found that NF-κB target genes involved in proliferation were upregulated in CD40-activated DBC1 KO B cells. In agreement with our previous findings, we observed increased expression of the NF-κB target genes CCNB1, CDC20, and BIRC5 in DBC1 KO B cells (Fig. 5E). However, RelB μ B cells have basal expression of all three genes, which could not be rescued by the DKO B cells (Fig. 5E). In sum, these experiments show that DBC1 negatively regulates B cell proliferation, Ig production, and NF-κB target gene expression through a RelB-dependent mechanism.

FIGURE 5.
FIGURE 5.

Increased proliferation and Ig production in DBC1 KO B cells is abrogated by deletion of RelB. (A) Splenic B cells were isolated from WT, DBC1 KO, RelBshep/shep (RelB μ), and Dbc1−/− RelBshep/shep (DKO) mice. CFSE-stained B cells were stimulated with anti-CD40 or LPS+ BAFF for 5 d then subjected to flow cytometry for detection of proliferation. (B) Mean fluorescence intensity (MFI) of CFSE from (A). Reduction of MFI indicates increased proliferation. (C) Division index calculated from CFSE experiments as performed in (A). (D) ELISA measuring IgG1 and IgA levels in culture supernatants of WT, DBC1 KO, RelB μ, and DKO B cells stimulated as in (A). (E) Real-time quantitative PCR detection of NF-κB target genes from WT, DBC1 KO, RelB μ, and DKO B cells activated overnight with anti-CD40. n = 5; error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001. n.s., not significant.

DBC1 interacts with IKKα and IKKβ in B cells

Through a mass spectrometry screen, DBC1 was previously identified as one of the proteins that interact with the IKKβ (22). IKKs are key components of the NF-κB regulatory complex, and they can activate NF-κB by phosphorylating IĸBs to promote its degradation (18). However, unlike IKKβ, which regulates classical NF-κB signaling through phosphorylation of IĸBs, IKKα is required for alternative NF-κB signaling by promoting processing of p100 to active p52 (1, 21). Based on published literature and our Co-IP studies, we hypothesized that DBC1 could be regulated by upstream components of NF-κB signaling (i.e., the IKK regulatory complex). We found that in addition to IKKβ, DBC1 interacts with greater affinity to the alternative NF-κB regulator IKKα, both in HEK293T cells (Fig. 6A, 6B) and in primary B cells (Fig 6C, 6D). Interestingly, DBC1 interaction with IKKα and IKKβ is reduced in B cells upon stimulation (Fig. 6C, 6D), suggesting that CD40 stimulation triggers the release of DBC1 from the IKK regulatory complex. Together with our observation that DBC1 suppresses the alternative NF-κB pathway, these results suggest that the regulatory function of DBC1 is regulated by IKKα.

FIGURE 6.
FIGURE 6.

DBC1 interacts with IKKα and IKKβ and is serine phosphorylated by IKKα. (A) Flag-tagged IKKα and IKKβ were each cotransfected with Myc-tagged DBC1, and cell lysates were immunoprecipitated with anti-Flag Ab. Interaction with DBC1 was detected by anti-Myc Ab. (B) Densitometry analysis of IKKα and IKKβ precipitates relative to IKKα in lane 2. (C) Primary B cells from WT mice were activated with the indicated stimuli for 1 h. Cell lysates were then immunoprecipitated with anti-DBC1 Ab, and interactions with IKKα and IKKβ were detected by immunoblotting with Ab recognizing both IKKα and IKKβ. (D) Densitometry analysis of IKKα and IKKβ protein in the precipitates relative to IKKα levels at the naive state. (E) Myc-tagged DBC1 was cotransfected with Flag-tagged IKKα and IKKβ into HEK293T, cell lysates were then immunoprecipitated with anti-Myc, and phosphorylation of DBC1 was detected using anti-phosphoSerine (α-pS) and anti-phosphoThreonine (α-pT) Abs. (F) Densitometry analysis of pS and pT levels from (E) relative to lane 1 (overexpression of Myc-DBC1 alone). (G) Primary B cells were stimulated with α-CD40 for the indicated times, DBC1 was immunoprecipitated from cell lysates, and phosphorylation of DBC1 was detected using α-pS Ab. Normal rabbit IgG serves as negative control, and whole cell lysate (WCL) as loading control. (H) Densitometry analysis of p-DBC1 as detected in (G) are normalized to lane 2 (naive state). n = 5; error bars represent SEM. *p < 0.05, ***p < 0.0001.

DBC1 is serine-phosphorylated by IKKα

Because IKKs function as serine/threonine kinases, we speculated that DBC1 could be phosphorylation substrates of IKKs. Indeed, using anti-phosphorylated Serine (α-pS), but not anti-phosphorylated Threonine (α-pT), Abs, we detected weak phosphorylation of DBC1, which was further enhanced by overexpression of either IKKα or IKKβ (Fig. 6E, 6F). In agreement with our observation that IKKα interacts with greater affinity with DBC1 than IKKβ, IKKα expression induced higher levels of DBC1 serine phosphorylation (Fig. 6E, 6F). Similarly, we detected phosphorylated serine residues on DBC1 in primary B cells (Fig. 6G, 6H). Interestingly, DBC1 phosphorylation was reduced 1 h upon CD40 stimulation, similar to its interaction with IKKα/β, and further reduced at 16 h upon stimulation. These results suggest that IKK, in particular IKKα, is a serine kinase of DBC1.

Serine phosphorylation of DBC1 at its C terminus is required for interaction with IKKα and RelB

DBC1 was found through mass spectrometry to have a cluster of phosphorylatable serine residues at the C terminus using PhosphoSitePlus (36). Indeed, replacing all six serine residues at the C terminus of DBC1 with alanine (DBC1-SA) abrogated serine phosphorylation of DBC1 (Fig. 7A). To elucidate the functional consequence of IKK-mediated DBC1 phosphorylation, we compared the ability of WT and phosphorylation-deficient DBC1-SA mutant to interact with RelB. Indeed, cotransfecting IKKα enhanced the interaction between DBC1 and RelB. In contrast, the interaction between DBC1 and RelB and IKKα was largely diminished when the serine residues on DBC1 were replaced with alanine, indicating that phosphorylation at the C terminus serine cluster of DBC1 is required for its interaction with RelB (Fig. 7B, 7C). Therefore, based on our Co-IP experiments, we conclude that DBC1 interaction with RelB is regulated by the alternative NF-κB regulatory protein IKKα, and phosphorylation by IKKα promotes the interaction between DBC1 and IKKα, as well as DBC1 and RelB.

FIGURE 7.
FIGURE 7.

DBC1 Serine phosphorylation by IKKα is required for interaction with RelB and IKKα. (A) Myc-tagged DBC1 and Myc-tagged DBC1-SA mutant were each cotransfected with Flag-tagged IKKα. Both myc-DBC1 and myc-DBC1-SA were then immunoprecipitated using anti-Myc, and phosphorylation levels were detected using α-pS Ab. Densitometry analysis of pS levels in the precipitate (bottom bar graph) is shown; pS-levels are relative to lane 1 (overexpression of WT DBC1 alone). (B) Myc-tagged DBC1 or DBC1-SA mutant was cotransfected with Flag-tagged RelB with or without flag-tagged IKKα. Cell lysates were then immunoprecipitated with anti-Myc Ab. Interaction between DBC1 and RelB or IKKα were detected by immunoblotting for Flag. (C) Densitometry analysis of precipitated RelB from (B) relative lane 2 (overexpression of WT DBC1 and RelB). (D) Schematic of proposed molecular mechanism of DBC1 suppression of B cells. (Di) DBC1 interacts with both IKKα and RelB in B cells and maintains the balance of active and inactive NF-κB. (Dii) Loss of DBC1 suppression of RelB leads to imbalanced increase of alternative NF-κB activity, leading to increased B cell proliferation and Ig production. n = 5; error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

In summary, our data indicate that DBC1 is a crucial regulator of B cell activation and Ig production by suppressing RelB activity. First, the loss of DBC1 in mice leads to a spontaneous increase in Ig production in the serum at 10 mo of age. Second, we determined that DBC1 selectively interacts with alternative NF-κB members RelB and p52, and the LZ domain of DBC1 is required and sufficient for this interaction. Third, the increased proliferation and Ig production in DBC1KO B cells is abrogated by deletion of RelB. Fourth, DBC1 interacts with both IKKα and IKKβ, with significantly higher affinity with the former. Lastly, we found that DBC1 is serine phosphorylated by IKKα, and its phosphorylation is required for its interaction with both RelB and IKKα. We previously identified a novel function of DBC1 as a suppressor of B cell activation. Furthering our previous work, we now determine that DBC1 suppresses B cell activation through RelB, and identify a novel regulatory mechanism of DBC1 through its interaction with IKKα and RelB.

Discussion

In this study, we show that loss of DBC1 leads to spontaneous overproduction of Ig in mice at 10 mo of age, and we confirm that DBC1 suppresses B cell activation through RelB using a double-KO genetic model. In terms of the molecular mechanism of DBC1, we have shown that DBC1 preferentially interacts with alternative NF-κB members RelB and p52, and the LZ domain is necessary and sufficient for its interaction. In addition, DBC1 interacts with the NF-κB regulators IKKα and IKKβ. Lastly, DBC1 is serine phosphorylated by IKKα, and its phosphorylation regulates its interaction with both IKKα and RelB. Based on our findings, we propose that DBC1 regulates B cell activation by binding to RelB:p52 as well as the IKK regulatory complex. Upon B cell stimulation, loss of DBC1 phosphorylation, and its release from IKKα and RelB, allows for full RelB-mediated B cell activation. In WT B cells, through IKKα-mediated phosphorylation, DBC1 maintains the balance between active and inactive alternative NF-κB. Loss of DBC1 function or regulation in B cells thus leads to increased RelB activity, hyperproliferation, and increased Ig production in mice (Fig. 7D).

Loss of integrity of B cell regulation and polyclonal expansion of autoreactive B cells especially increases with age (37–39). In our experimental model, the loss of DBC1 leads to the production of self-reactive Abs spontaneously with age. The alternative NF-κB signaling pathway, which is induced by CD40L and BAFF in B cells, plays a central role in peripheral B cell regulation (15, 40–43). Specifically, increased BAFF levels or overexpression of CD40 or BAFF are associated with B cell–mediated autoimmunity (14, 16, 43–45). Based on its suppressive function on the alternative NF-κB pathway, it is possible that the loss of DBC1 in mice leads to increased CD40 and BAFFR-induced alternative NF-κB signaling, resulting in the breakdown of B cell regulation and the presence of autoantibodies. The alternative NF-κB signaling pathway has also been shown to be important in IgG and IgA class switching (12, 46, 47), consistent with our observation that IgG and IgA isotypes are increased in the sera of aged DBC1 KO mice. However, we did not detect spontaneous onset of clinical symptoms of autoimmunity such as glomerulonephritis in DBC1-deficient mice (data not shown). It is possible that an additional genetic predisposition or antigenic challenge is required to induce additional autoimmune symptoms. In support of our speculation, we have previously shown that in an experimental autoimmune myasthenia gravis setting, DBC1-deficient mice have elevated anti-acetylcholine Ab, and exhibit increased disease severity as a result (34).

Using a microarray approach, we previously identified NF-κB target genes to be dysregulated by loss of DBC1. ChIP experiments also show that the activity of alternative NF-κB member RelB in particular was increased in DBC1 KO B cells (34). Because NF-κB plays a critical role in proliferation and isotype switching of activated B cells, we proposed that the hyperproliferative and increased IgG and IgA phenotype in DBC1 KO mice is dependent on increased RelB activity. To confirm that loss of DBC1 leads to increased B cell activation through RelB, we used a Dbc1−/− RelBshep/shep double mutant strain and tested for B cell activation and function. Consistent with our hypothesis, loss of RelB function abrogated the hyperproliferative phenotype of DBC1 KO mice, affirming that DBC1 suppresses B cell proliferation and Ig production by inhibiting RelB function. Furthermore, through our Co-IP experiments, we show that DBC1 preferentially interacts with RelB and p52, but not other NF-κB members RelA, cRel, and p50, consistent with our hypothesis that DBC1 suppresses B cell activation through the alternative NF-κB pathway.

NF-κB activity is regulated by the IKK regulatory complex (18). Although IKKα and IKKβ are found within the same complex in vivo, genetic studies have shown that IKKα and IKKβ regulate alternative and classical NF-κB activity differentially (18, 48). DBC1 was previously identified as an interacting protein with IKKβ through a mass spectrometry screen (22). In our study, we confirmed that in addition to RelB:p52, DBC1 interacts with IKKα and IKKβ in B cells. Moreover, in agreement with our finding that DBC1 regulates the alternative NF-κB pathway, DBC1 interacts with a much higher affinity with IKKα, which is required for regulation of alternative NF-κB signaling, rather than IKKβ (1, 21). Similarly, IKKα is more effective in increasing serine phosphorylation of DBC1 compared with IKKβ. Interestingly, upon CD40 activation in B cells, DBC1 interaction with IKKα and IKKβ is reduced, and it correlates with reduced DBC1 phosphorylation, especially after 16 h. Moreover, in primary B cells, loss of DBC1 phosphorylation at 16 h leads to reduced interaction between DBC1 and RelB and a shift from its interaction with p52 to its inactive precursor p100 (Fig. 2B). On the basis of our data, we propose that DBC1 phosphorylation by IKKα enables DBC1 suppression of RelB, and the later loss of phosphorylation on DBC1 reduces its suppressive function, leading to full RelB activity. In support of this proposition, replacing six serines on DBC1 at its C terminus reduced phosphorylation levels of DBC1 and its interaction with RelB. Basal IKK kinase activity has been reported to serve important metabolic functions, and it is actively maintained during cell homeostasis (49–51). Our data thus suggest that in addition to promoting the activation of alternative NF-κB by phosphorylating p100 for processing, IKKα also negatively regulates alternative NF-κB activity by phosphorylating DBC1 and maintaining its suppression of RelB:p52 activity. In addition, DBC1 was reported to be phosphorylated at T454 by ataxia telangiectasia mutant kinase during the DNA damage response (52). In our experiments, using α-pT Ab, we could not detect any threonine phosphorylation of DBC1. Instead, serine phosphorylation regulates DBC1 function on the alternative NF-κB pathway. Therefore, DBC1 appears to regulate B cell activation independent of DNA damage responses.

Thus, our results further define the role of DBC1 as an endogenous suppressor of B cell proliferation and Ig production, as the loss of DBC1 in mice leads to spontaneous production of autoreactive Ig at 10 mo of age. In addition, using a double mutant genetic model, we confirm that DBC1 regulates B cell activation by suppressing the alternative NF-κB pathway. At a molecular level, DBC1 suppresses B cell activation selectively binding to alternative NF-κB members RelB and p52. In addition, DBC1 preferentially binds to IKKα and is serine-phosphorylated at the C terminus. Lastly, phosphorylation of DBC1 by IKKα is crucial for its interaction with IKKα and RelB. DBC1 has been implicated in the regulation of proliferation in solid tumors, but the study of DBC1 in the immune response has been limited. As the alternative NF-κB pathway is a crucial regulator of B cell activation and autoimmunity, defining the role of DBC1 in regulating the alternative NF-κB pathway provides a rationale for further study of its role in the hematopoietic system, in B cell Ig production, and in autoimmune regulation.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by National Institutes of Health Grants AI079056 and DK083050 (and their supplemental grants to D.F.), 1R01AI107516-01A1 (to B.S.P.), and DK-084055/DK/NIDDK NIH HHS/United States (to E.C.).

  • Abbreviations used in this article:

    ChIP
    chromatin immunoprecipitation
    CO-Ip
    coimmunoprecipitation
    DBC1
    deleted in breast cancer 1
    DKO
    RelB/DBC1 double KO
    IKK
    inhibitor of κ B kinase
    KO
    knockout
    LZ
    leucine zipper
    RT
    room temperature
    WT
    wild type.

  • Received April 2, 2015.
  • Accepted August 13, 2015.

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