Alternative NFB signaling is crucial for B cell activation and immunoglobulin

Alternative NFB signaling is crucial for B cell activation and immunoglobulin production, and is mainly regulated by the IKK regulatory complex. by IKK. Our results define the molecular mechanism of DBC1 inhibition of alternative NFkB activation in suppressing B cell activation. Introduction The NFB pathway is critical in many processes such as cell survival, inflammatory cytokine signaling and apoptosis (1C3). While the canonical signaling pathway is highly expressed in most cell types, 837422-57-8 the alternative NFB pathway is central to a much smaller subset of cell types, namely bone, dendritic cells and B cells (1, 837422-57-8 4C6). In addition, the canonical and alternative NFB pathways have distinct functions (3). In B cells, the alternative NFB pathway regulates development from transitional to mature B cells, cell cycle entry, isotype switching and differentiation, and plasma cell survival (5, 7C13). As a result, either reduced or increased alternative NFB activation is associated with defective B cell response or B cell-mediated autoimmune diseases respectively (5, 12, 14C17). One of the main regulatory mechanisms of the NFB pathway is through the Inhibitor of kappa B Kinase (IKK) regulatory complex, which consists of at least IKK, IKK and or IKK (NEMO) (18). IKK and IKK are both about 750 amino acids in size, whereas IKK is 300 amino acids smaller in size, and has a distinct structure from the former two (18, 19). Although IKK and structurally very similar, knock out studies show that IKK and IKK differentially regulate the alternative and canonical NFB pathways respectively (18C21). IKK preferentially binds to and phosphorylates IB, which is then degraded to release RelA: p50 heterodimers into the Rabbit Polyclonal to C-RAF (phospho-Ser301) nucleus (18). On the other hand, IKK phosphorylates the regulatory domain of p100, a necessary step to yield transcriptionally active RelB:p52 dimers (18, 21). (DBC1) was found to interact with IKK through a mass spectrometry screen (22). Although initially identified as a tumor suppressor, the role of DBC1 is cell-type dependent, as upregulated DBC1 levels are found in various other cancers (23C26). In addition to IKK, DBC1 has been shown to interact with various nuclear proteins, such as ER, AR, BRCA1, HDAC3, SUV39H1, and Sirt1, thus implicating its 837422-57-8 role in regulating transcription and epigenetic modification (27C33). 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 further report that loss of DBC1 in mice leads to spontaneous dysregulation of B cells at 10 months of age, leading to increased production of autoreactive immunoglobulin. 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 NFB members RelB and p52, as well as its regulator IKK. Lastly, while the N-terminus Leucine Zipper (LZ) domain of DBC1 is required for its interaction with RelB, phosphorylation of DBC1 at its C terminus by IKK is required for its interaction with both RelB and IKK Our study further defines the molecular mechanism of DBC1 suppression of NFB, and its role in B cell regulation. Materials and Methods Mouse and Cell Lines HEK293T and NIH3T3 cells lines were maintained in DMEM (Gibco) supplemented with 10% Fetal Calf Serum (FCS) and 1% Penicillin/Streptomycin. EL4 cell line was maintained in RPMI supplemented with 10% FCS and 1% penicillin/streptomycin. mice were gifted by the Chini lab, and further backcrossed to C57BL/6 background for more than 5 generations. 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, Antibodies 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 using 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-IKK, pCR-Flag-IKK plasmids were purchased from Addgene. Antibodies used for immunoblotting and co-immunoprecipitation were anti-p30/DBC1 (Bethyl laboratories), anti-RelB (C-19) (D-4), p100/p52 (C-5), Myc (A-14), (9E10), IKK/ (H-470) (Santa Cruz), anti-Flag (F7425) (F1804) (Sigma), anti-Tubulin (DM1A) (Calbiochem) anti-phosphoserine (AB1603), phosphothreonine (AB1607) (Millipore). Primary B cell Isolation and Culture Primary B cells were negatively isolated from 8C12 week old mice using Dynabeads? Mouse CD43 (Untouched B cells) (Life Technologies) 837422-57-8 per manufacturers instructions. Primary B cells were maintained at 106/mL in.