Marzenna Blonska

Ubiquitination of RIP Is Required for Tumor Necrosis Factor α-induced NF-κB Activation

Stimulation of cells with tumor necrosis factor (TNFα) triggers a recruitment of various signaling molecules, such as RIP, to the TNFαreceptor 1 complex, leading to activation of NF-κB. Previous studies indicate that RIP plays an essential role for TNFα-induced NF-κB activation, but the molecular mechanism by which RIP mediates TNFαsignals to activate NF-κB 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 TNFα-induced NF-κB activation. In this study, we have identified Lys377 of RIP as the functional ubiquitination site, because mutating this residue to arginine completely abolished RIP-mediated NF-κB activation. The K377R mutation of RIP cannot undergo ligand-dependent ubiquitination and fails to recruit its downstream signaling components into the TNFαreceptor 1 complex. Together, our studies provide the first genetic evidence that the ubiquitination of RIP is required for TNFα-induced NF-κB activation.

Inflammatory T Cell Responses Rely on Amino Acid Transporter ASCT2 Facilitation of Glutamine Uptake and mTORC1 Kinase Activation

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 naive 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.

Ubiquitination of RIP is required for TNFα-induced NF-κB activation

Stimulation of cells with tumor necrosis factor (TNFα) triggers a recruitment of various signaling molecules, such as RIP, to the TNFαreceptor 1 complex, leading to activation of NF-κB. Previous studies indicate that RIP plays an essential role for TNFα-induced NF-κB activation, but the molecular mechanism by which RIP mediates TNFαsignals to activate NF-κB 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 TNFα-induced NF-κB activation. In this study, we have identified Lys377 of RIP as the functional ubiquitination site, because mutating this residue to arginine completely abolished RIP-mediated NF-κB activation. The K377R mutation of RIP cannot undergo ligand-dependent ubiquitination and fails to recruit its downstream signaling components into the TNFαreceptor 1 complex. Together, our studies provide the first genetic evidence that the ubiquitination of RIP is required for TNFα-induced NF-κB activation.

NF-κB signaling pathways regulated by CARMA family of scaffold proteins

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.

TAK1 Is Recruited to the Tumor Necrosis Factor-α (TNF-α) Receptor 1 Complex in a Receptor-interacting Protein (RIP)-dependent Manner and Cooperates with MEKK3 Leading to NF-κB Activation

Receptor-interacting protein (RIP) plays a critical role in tumor necrosis factor-α (TNF-α)-induced IκB kinase (IKK) activation and subsequent activation of transcription factor NF-κB. However, the molecular mechanism by which RIP mediates TNF-α-induced NF-κB activation is not completely defined. In this study, we have found that TAK1 is recruited to the TNF-α receptor complex in a RIP-dependent manner following the stimulation of TNF-α receptor 1 (TNF-R1). Moreover, a forced recruitment of TAK1 to TNF-R1 in the absence of RIP is sufficient to mediate TNF-α-induced NF-κB activation, indicating that the major function of RIP is to recruit its downstream kinases to the TNF-R1 complex. Interestingly, we also find that TAK1 and MEKK3 form a functional complex, in which TAK1 regulates autophosphorylation of MEKK3. The TAK1-mediated regulation of MEKK3 phosphorylation is dependent on the kinase activity of TAK1. Although TAK1-MEKK3 interaction is not affected by overexpressed TAB1, TAB1 is required for TAK1 activation and subsequent MEKK3 phosphorylation. Together, we conclude that TAK1 is recruited to the TNF-R1 complex via RIP and likely cooperates with MEKK3 to activate NF-κB in TNF-α signaling.

Phosphorylation and ubiquitination of the IκB kinase complex by two distinct signaling pathways

The IκB kinase (IKK) complex serves as the master regulator for the activation of NF‐κB by various stimuli. It contains two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ/NEMO. The activation of IKK complex is dependent on the phosphorylation of IKKα/β at its activation loop and the K63‐linked ubiquitination of NEMO. However, the molecular mechanism by which these inducible modifications occur remains undefined. Here, we demonstrate that CARMA1, a key scaffold molecule, is essential to regulate NEMO ubiquitination upon T‐cell receptor (TCR) stimulation. However, the phosphorylation of IKKα/β activation loop is independent of CARMA1 or NEMO ubiquitination. Further, we provide evidence that TAK1 is activated and recruited to the synapses in a CARMA1‐independent manner and mediate IKKα/β phosphorylation. Thus, our study provides the biochemical and genetic evidence that phosphorylation of IKKα/β and ubiquitination of NEMO are regulated by two distinct pathways upon TCR stimulation.

CARMA1-mediated NF-κB and JNK activation in lymphocytes

Summary:  Activation of transcription factor nuclear factor‐κB (NF‐κB) and Jun N‐terminal kinase (JNK) play the pivotal roles in regulation of lymphocyte activation and proliferation. Deregulation of these signaling pathways leads to inappropriate immune response and contributes to the development of leukemia/lymphoma. The scaffold protein CARMA1 [caspase‐recruitment domain (CARD) membrane‐associated guanylate kinase (MAGUK) protein 1] has a central role in regulation of NF‐κB and the JNK2/c‐Jun complex in both B and T lymphocytes. During last several years, tremendous work has been done to reveal the mechanism by which CARMA1 and its signaling partners, B cell CLL‐lymphoma 10 and mucosa‐associated lymphoid tissue 1, are activated and mediate NF‐κB and JNK activation. In this review, we summarize our findings in revealing the roles of CARMA1 in the NF‐κB and JNK signaling pathways in the context of recent advances in this field.

USP18 inhibits NF-κB and NFAT activation during Th17 differentiation by deubiquitinating the TAK1–TAB1 complex

Ubiquitin-specific protease 18 inhibits ubiquitination of TAK1–TAB complexes to restrict IL-2 production and promote Th17 differentiation and autoimmune responses.

CARMA1 controls Th2 cell-specific cytokine expression through regulating JunB and GATA3 transcription factors

The scaffold protein CARMA1 is required for the TCR-induced lymphocyte activation. In this study, we show that CARMA1 also plays an essential role in T cell differentiation. We have found that the adoptive transfer of bone marrow cells expressing constitutively active CARMA1 results in lung inflammation, eosinophilia, and elevated levels of IL-4, IL-5, and IL-10 in recipient mice. In contrast, CARMA1-deficient T cells are defective in TCR-induced expression of Th2 cytokines, suggesting that CARMA1 preferentially directs Th2 differentiation. The impaired cytokine production is due to reduced expression of JunB and GATA3 transcription factors. CARMA1 deficiency affects JunB stability resulting in its enhanced ubiquitination and degradation. In contrast, TCR-dependent induction of GATA3 is suppressed at the transcriptional level. We also found that supplementation with IL-4 partially restored GATA3 expression in CARMA1-deficient CD4+ splenocytes and subsequently production of GATA3-dependent cytokines IL-5 and IL-13. Therefore, our work provides the mechanism by which CARMA1 regulates Th2 cell differentiation.

The cell cycle regulator 14-3-3σ opposes and reverses cancer metabolic reprogramming

Summary Extensive reprogramming of cellular energy metabolism is a hallmark of cancer. Despite its importance, the molecular mechanism controlling this tumour metabolic shift remains not fully understood. Here we show that 14-3-3σ regulates cancer metabolic reprogramming and protects cells from tumourigenic transformation. 14-3-3σ opposes tumour-promoting metabolic programs by enhancing c-Myc poly-ubiquitination and subsequent degradation. 14-3-3σ demonstrates the suppressive impact on cancer glycolysis, glutaminolysis, mitochondrial biogenesis and other major metabolic processes of tumours. Importantly, 14-3-3σ expression levels predict overall and recurrence-free survival rates, tumour glucose uptake and metabolic gene expression in breast cancer patients. Thus, these results highlight that 14-3-3σ is an important regulator of tumour metabolism, and loss of 14-3-3σ expression is critical for cancer metabolic reprogramming. We anticipate that pharmacologically elevating the function of 14-3-3σ in tumours could be a promising direction for targeted anti-cancer metabolism therapy development in future.