TRAF3 as a Multifaceted Regulator of B Lymphocyte Survival and Activation

The adaptor protein TNF receptor-associated factor 3 (TRAF3) serves as a powerful negative regulator in multiple aspects of B cell biology. Early in vitro studies in transformed cell lines suggested the potential of TRAF3 to inhibit signaling by its first identified binding receptor, CD40. However, because the canonical TRAF3 binding site on many receptors also mediates binding of other TRAFs, and whole-mouse TRAF3 deficiency is neonatally lethal, an accurate understanding of TRAF3's specific functions was delayed until conditional TRAF3-deficient mice were produced. Studies of B cell-specific TRAF3-deficient mice, complemented by investigations in normal and malignant mouse and human B cells, reveal that TRAF3 has powerful regulatory roles that are unique to this TRAF, as well as functions context-specific to the B cell. This review summarizes the current state of knowledge of these roles and functions. These include inhibition of signaling by plasma membrane receptors, negative regulation of intracellular receptors, and restraint of cytoplasmic NF- κB pathways. TRAF3 is also now known to function as a resident nuclear protein, and to impact B cell metabolism. Through these and additional mechanisms TRAF3 exerts powerful restraint upon B cell survival and activation. It is thus perhaps not surprising that TRAF3 has been revealed as an important tumor suppressor in B cells. The many and varied functions of TRAF3 in B cells, and new directions to pursue in future studies, are summarized and discussed here

HOIL-1L Interacting Protein (HOIP) as an NF-κB Regulating Component of the CD40 Signaling Complex

The tumor necrosis factor receptor (TNFR) superfamily mediates signals critical for regulation of the immune system. One family member, CD40, is important for the efficient activation of antibody-producing B cells and other antigen-presenting cells. The molecules and mechanisms that mediate CD40 signaling are only partially characterized. Proteins known to interact with the cytoplasmic domain of CD40 include members of the TNF receptor-associated factor (TRAF) family, which regulate signaling and serve as links to other signaling molecules. To identify additional proteins important for CD40 signaling, we used a combined stimulation/immunoprecipitation procedure to isolate CD40 signaling complexes from B cells and characterized the associated proteins by mass spectrometry. In addition to known CD40-interacting proteins, we detected SMAC/DIABLO, HTRA2/Omi, and HOIP/RNF31/PAUL/ZIBRA. We found that these previously unknown CD40-interacting partners were recruited in a TRAF2-dependent manner. HOIP is a ubiquitin ligase capable of mediating NF-κB activation through the ubiquitin-dependent activation of IKKγ. We found that a mutant HOIP molecule engineered to lack ubiquitin ligase activity inhibited the CD40-mediated activation of NF-κB. Together, our results demonstrate a powerful approach for the identification of signaling molecules associated with cell surface receptors and indicate an important role for the ubiquitin ligase activity of HOIP in proximal CD40 signaling

A BAFF-R mutation associated with non-Hodgkin lymphoma alters TRAF recruitment and reveals new insights into BAFF-R signaling

A BAFF receptor mutation associated with non-Hodgkin lymphoma provides new insight into the proximal players of normal BAFF-R signaling

IKK recruitment to the CD40 signaling complex is defective in HOIP-deficient cells.

<p>(A) SMAC peptide treatment reduces recruitment of cIAP1 to CD40 and may modify HOIP recruitment. A20.2J cells were incubated for six hours with membrane-permeable SMAC-N7 peptide or 1.5% DMSO (solvent used for the peptide). Following the incubation, cell lysates were prepared, fractionated by SDS-PAGE, and evaluated by Western blot (lanes 1 and 2). Cells incubated with DMSO or SMAC-N7 were also stimulated with magnetic beads coated with anti-CD40 or an isotype control antibody. Immunoprecipitated (IP) material bound to the beads was loaded in lanes 3-5. Samples of the cell lysates after immunoprecipitation appear in lanes 6–8. Western blots were probed with antibodies specific for cIAP1, TRAF2, TRAF3, and HOIP (approximate molecular weights indicated on left). Similar results were obtained in two additional experiments. (B) CD40 was isolated by immunoprecipitation (as in (A)) from A20.2J cells and HOIP-deficient cells transduced with an empty retroviral vector (pMIP) or a retroviral vector encoding HOIP. Material immunoprecipitated with an isotype control antibody (isotype) or anti-CD40 antibody was examined by Western blotting for CD40, TRAF2, TRAF3, cIAP1, HOIP, IKKα/β, and IKKγ (right panels). HOIP expression was required for coprecipitation of IKK proteins with CD40. Cell lysates from unstimulated cells are shown in the left panels. Similar results were obtained in a second experiment and in two experiments with a second HOIP-deficient clone. (C) To further evaluate HOIP-dependent recruitment of IKKγ to CD40, A20.2J cells or HOIP-deficient (HOIP^-/-) A20.2J cells were transduced with an empty retroviral vector or a retroviral construct encoding BP epitope-tagged IKKγ (noted in the figure as pMIP and IKKγ, respectively). Lysates (lanes 1–3) and immunoprecipitation (IP) samples (lanes 4–11) from the cell lines were fractionated by SDS-PAGE and evaluated by Western blotting with antibodies to the BP tag (IKKγ, upper panel) and TRAF2. The anti-CD40 IP sample in lane 11 was treated with λ phosphatase; the sample in lane 10 was mock-treated. Protein samples (minus those treated with phosphatase) were also fractionated on a separate gel (lower acrylamide concentration) for the evaluation of TRAF3 and HOIP (bottom two panels). Similar results were obtained in two additional experiments.</p