Glutamine has been implicated as an immunomodulatory nutrient, but how glutamine uptake is mediated during T-cell activation is poorly understood. We have shown that naïve T-cell activation is coupled with rapid glutamine uptake, which depended on the amino acid transporter ASCT2. ASCT2 deficiency impaired the induction of T helper-1 (Th1) and Th17 cells and attenuated inflammatory T-cell responses in mouse models of immunity and autoimmunity. Mechanistically, ASCT2 was required for T cell receptor (TCR)-stimulated activation of the metabolic kinase mTORC1. We have further shown that TCR-stimulated glutamine uptake and mTORC1 activation also required a TCR signaling complex composed of the scaffold protein CARMA1, the adaptor molecule BCL10, and the paracaspase MALT1. This function was independent of IKK kinase, a major downstream target of the CARMA1 complex. These findings highlight a mechanism of T-cell activation involving ASCT2-dependent integration of the TCR signal and a metabolic signaling pathway.

CARMA1 mediates T cell receptor (TCR)-induced NF-kappaB activation. However, how TCR links to CARMA1 in the signaling pathway is not clear. Here, we show that CARMA1 is inducibly phosphorylated after TCR-CD28 costimulation. This phosphorylation is likely induced by PKCtheta, since PKCtheta induces phosphorylation of CARMA1 in vitro and in vivo. Our results indicate that the PKCtheta-induced phosphorylation of CARMA1 likely occurs on Ser552 on the Linker region of CARMA1. Importantly, expression of CARMA1 mutant, in which Ser552 is mutated, fails to mediate TCR-induced NF-kappaB activation in CARMA1-deficient T cells. The functional defect of this CARMA1 mutant is likely due to the fact that this mutant cannot be phosphorylated at the critical residue, thereby failing to recruit the downstream signaling components into the immunological synapse. Together, our studies provide the first genetic evidence that the phosphorylation of CARMA1 plays a critical role in the TCR signaling pathway.

Stimulation of cells with tumor necrosis factor (TNF␣) triggers a recruitment of various signaling molecules, such as RIP, to the TNF␣ receptor 1 complex, leading to activation of NF-B. Previous studies indicate that RIP plays an essential role for TNF␣-induced NF-B activation, but the molecular mechanism by which RIP mediates TNF␣ signals to activate NF-B is not fully defined. Earlier studies suggest that RIP undergoes a ligand-dependent ubiquitination. However, it remains to be determined whether the ubiquitination of RIP is required for TNF␣-induced NF-B activation. In this study, we have identified Lys 377 of RIP as the functional ubiquitination site, because mutating this residue to arginine completely abolished RIP-mediated NF-B activation. The K377R mutation of RIP cannot undergo ligand-dependent ubiquitination and fails to recruit its downstream signaling components into the TNF␣ receptor 1 complex. Together, our studies provide the first genetic evidence that the ubiquitination of RIP is required for TNF␣-induced NF-B activation.NF-B is a homo-or heterodimeric transcription factor that controls the expression of various genes involved in inflammatory, apoptotic, and immune responses. In resting cells, the activity of NF-B is controlled through its cytoplasmic sequestration by a family of inhibitors, IB (Inhibitor of NF-B) (1). In response to extracellular stimuli, IB proteins are phosphorylated by the IB kinase (IKK) 2 complex, then ubiquitinated, and rapidly degraded, which leads to the nuclear localization and activation of NF-B (2). One of the most potent NF-B activators is the proinflammatory cytokine, tumor necrosis factor (TNF)␣. TNF␣ functions through the TNF receptors, mainly TNFR1, on the cell surface. The binding of TNF␣ to TNFR1 causes trimerization of the receptor and recruitment of the adaptor protein TRADD (TNF receptor-associated death domain) (3). TRADD further recruits TARF2 (TNF receptor-associated factor 2) (4), FADD (Fas-associated death domain) (4), and RIP (receptor-interacting protein) (5, 6).RIP is a serine/threonine kinase that plays an essential role in TNF␣-induced NF-B activation (6, 7). It contains an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain. Earlier studies indicate that the RIP kinase domain is dispensable for the activation of NF-B (6, 8), whereas its death domain is required for the association with the upstream signaling component TRADD (5). The intermediate domain is required for RIP-mediated NF-B activation (6) and plays an important role for interacting with its downstream signaling components such as NEMO, the regulatory subunit of the IKK complex, and other molecules (9). Therefore, RIP likely functions as an adaptor molecule to mediate the TNF␣ signaling cascade. Although genetic studies have demonstrated that TAK1 (tumor growth factor ␤-activated kinase 1) and MEKK3 are involved in mediating TNF␣-induced NF-B activation downstream of RIP (10 -15), the mechanism by which RIP activates these downstream kinases is not fully def...

The NF-κB family of transcription factors plays a crucial role in cell activation, survival and proliferation. Its aberrant activity results in cancer, immunodeficiency or autoimmune disorders. Over the past two decades, tremendous progress has been made in our understanding of the signals that regulate NF-κB activation, especially how scaffold proteins link different receptors to the NF-κB-activating complex, the IκB kinase complex. The growing number of these scaffolds underscores the complexity of the signaling networks in different cell types. In this review, we discuss the role of scaffold molecules in signaling cascades induced by stimulation of antigen receptors, G-protein-coupled receptors and C-type Lectin receptors, resulting in NF-κB activation. Especially, we focus on the family of Caspase recruitment domain (CARD)-containing proteins known as CARMA and their function in activation of NF-κB, as well as the link of these scaffolds to the development of various neoplastic diseases through regulation of NF-κB.

G protein-coupled receptors (GPCRs) play pivotal roles in regulating various cellular functions. Although many GPCRs induce NF-B activation, the molecular mechanism of GPCR-induced NF-B activation remains largely unknown. CARMA3 (CARD and MAGUK domain-containing protein 3) is a scaffold molecule with unknown biological functions. By generating CARMA3 knockout mice using the gene targeting approach, here we show CARMA3 is required for GPCR-induced NF-B activation. Mechanistically, we found that CARMA3 deficiency impairs GPCR-induced IB kinase (IKK) activation, although it does not affect GPCR-induced IKK␣/␤ phosphorylation, indicating that inducible phosphorylation of IKK␣/␤ alone is not sufficient to induce its kinase activity. We also found that CARMA3 is physically associated with NEMO/IKK␥, and induces polyubiquitination of an unknown protein(s) that associates with NEMO, likely by linking NEMO to TRAF6. Consistently, we found TRAF6 deficiency also abrogates GPCR-induced NF-B activation. Together, our results provide the genetic evidence that CARMA3 is required for GPCR-induced NF-B activation.[Keywords: NF-B; GPCR; CARMA3; neural tube] Supplemental material is available at http://www.genesdev.org.