Hajir Dadgostar

IRF3 Mediates a TLR3/TLR4-Specific Antiviral Gene Program

We have identified a subset of genes that is specifically induced by stimulation of TLR3 or TLR4 but not by TLR2 or TLR9. Further gene expression analyses established that upregulation of several primary response genes was dependent on NF-kappaB, commonly activated by several TLRs, and interferon regulatory factor 3 (IRF3), which was found to confer TLR3/TLR4 specificity. Also identified was a group of secondary response genes which are part of an autocrine/paracrine loop activated by the primary response gene product, interferon beta (IFNbeta). Selective activation of the TLR3/TLR4-IRF3 pathway potently inhibited viral replication. These results suggest that TLR3 and TLR4 have evolutionarily diverged from other TLRs to activate IRF3, which mediates a specific gene program responsible for innate antiviral responses.

Cooperation of multiple signaling pathways in CD40-regulated gene expression in B lymphocytes

CD40/CD40L interaction is essential for multiple biological events in T dependent humoral immune responses, including B cell survival and proliferation, germinal center and memory B cell formation, and antibody isotype switching and affinity maturation. By using high-density microarrays, we examined gene expression in primary mouse B lymphocytes after multiple time points of CD40L stimulation. In addition to genes involved in cell survival and growth, which are also induced by other mitogens such as lipopolysaccharide, CD40L specifically activated genes involved in germinal center formation and T cell costimulatory molecules that facilitate T dependent humoral immunity. Next, by examining the roles of individual CD40-activated signal transduction pathways, we dissected the overall CD40-mediated response into genes independently regulated by the individual pathways or collectively by all pathways. We also found that gene down-regulation is a significant part of the overall response and that the p38 pathway plays an important role in this process, whereas the NF-κB pathway is important for the up-regulation of primary response genes. Our finding of overlapping independent control of gene expression modules by different pathways suggests, in principle, that distinct biological behaviors that depend on distinct gene expression subsets can be manipulated by targeting specific signaling pathways.

An Intact Zinc Ring Finger Is Required for Tumor Necrosis Factor Receptor-associated Factor-mediated Nuclear Factor-κB Activation but Is Dispensable for c-Jun N-terminal Kinase Signaling

The diverse biological effects of the tumor necrosis factor (TNF) receptor superfamily are believed to be mediated in part through TNF receptor-associated factors (TRAFs), a family of cytoplasmic adaptor proteins which can activate intracellular signaling pathways, including the nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathways. TRAFs 2, 5, and 6 strongly activate both pathways when overexpressed; however, TRAF 3 (a close homologue of TRAF 5) does not significantly activate either pathway. The current study addresses the structural basis for this difference by substituting corresponding domains of TRAF 5 into TRAF 3 and testing activation of both pathways. A small region of TRAF 5 (the first zinc finger and 10 residues of the second zinc finger) is sufficient to convert TRAF 3 into an activator of both pathways. Also, an intact zinc ring finger is required for NF-κB activation but not JNK activation. In agreement with this finding, TRAF 2A, a TRAF 2 splice variant with an altered ring finger, is a specific activator of JNK. These findings suggest that different domains of TRAFs may be involved in NF-κB and JNK signaling. Also, alternative splicing of TRAFs may represent a novel mechanism whereby TNF family receptors can mediate distinct downstream effects in different tissues.

Membrane Localization of TRAF 3 Enables JNK Activation

Members of the tumor necrosis factor receptor family as well as other receptors achieve their diverse biological effects through the activation of intracellular signals including the c-Jun N-terminal kinase (JNK) pathway. Such signals are believed to be delivered through mediators known as TNF receptor-associated factors (TRAFs). Although the N-terminal zinc finger region of TRAFs has been shown to be essential for downstream signaling, there is no indication yet as to the nature of its role or of the factors that distinguish the N terminus of TRAF 3, which does not activate JNK in the systems examined thus far, from those of other TRAFs, which do activate this pathway. In the present study, it is shown that, among the known TRAFs, localization to the insoluble cell pellet fraction consistently correlates with JNK activation and that both characteristics map to the TRAF N terminus. Furthermore, it is demonstrated that forced localization of TRAF 3 to the cell membrane is sufficient to convert this molecule into an activator of JNK. This suggests that one of the roles of the TRAF N terminus may be to participate in interactions that promote the recruitment of TRAFs to the membrane and that this localization effect plays an important role in TRAF-mediated JNK activation.

T3JAM, a novel protein that specifically interacts with TRAF3 and promotes the activation of JNK1

Previous studies suggest that localization of tumor necrosis factor receptor (TNFR)‐associated factor (TRAF) family members is important for regulating their signal transduction. During a screen for TRAF3‐associated proteins that potentially alter TRAF3 subcellular localization and enable signal transduction, we identified a novel protein, T3JAM (TRAF3‐interacting Jun N‐terminal kinase (JNK)‐activating modulator). This protein associates specifically with TRAF3 but not other TRAF family members. Coexpression of T3JAM with TRAF3 recruits TRAF3 to the detergent‐insoluble fraction. More importantly, T3JAM and TRAF3 synergistically activate JNK but not nuclear factor (NF)‐κB. Our studies indicate that T3JAM may function as an adapter molecule that specifically regulates TRAF3‐mediated JNK activation.