Proinflammatory activation of NF-B requires an upstream kinase complex (IB-kinase; IKK) composed of two catalytic subunits (IKK␣ and IKK␤) and a noncatalytic regulatory component named NEMO (NF-B essential modulator). NEMO interacts with a COOH-terminal sequence within both IKKs termed the NEMO-binding domain (NBD), and a cell-permeable NBD peptide blocks NEMO/IKK␤ interactions and inhibits tumor necrosis factor-␣-induced NF-B. We report here that a peptide encompassing the NBD not only blocked association of both IKKs with NEMO but also disrupted preformed NEMO/IKK complexes in vitro. Furthermore, peptide blocking and alanine-scanning mutation studies revealed differences between the NBDs of IKK␣ and IKK␤, and mutational analysis of the IKK␤ NBD identified the physical properties required at each position to maintain association with NEMO. Finally, we demonstrate that loss of NEMO-binding by IKK␤ through deletion of the NBD renders it catalytically active and that potential phosphorylation within the IKK␤ NBD may serve as a signal to down-regulate IKK activity. Our findings therefore provide critical insight into the physical properties of the NBD that will be valuable for the design of drugs aimed at disrupting the IKK complex and also reveal potential regulatory mechanisms controlling the function of the IKK complex.

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RelB is an unusual member of the NF-〉 transcription factor family that acts as both a transcriptional activator as well as a repressor of NF-〉-dependent gene expression. Although RelB promotes gene expression when it associates with p50/NF-〉1 or p52/NF-〉2, the precise molecular mechanisms through which it represses NF-〉 remain unclear. To examine this inhibitory function in more detail, we employed reporter gene assays and found that RelB represses at the level of RelA. Furthermore, electrophoretic mobility shift analysis revealed that in vitro translated RelB impaired the DNA binding activity of RelA and that overexpressed RelB significantly reduced tumor necrosis factor-␣-induced RelA activity in murine embryonic fibroblasts. Intriguingly, this inhibitory effect was due to the formation of RelA⅐RelB heterodimers that were unable to bind to B sites in vitro strongly suggesting that these newly described NF-〉 dimers cannot bind DNA. Expression pattern analysis revealed that RelA⅐RelB heterodimers appeared at relatively low levels in both lymphoid and non-lymphoid cells. However, the presence of these complexes increased following stimulation with phorbolesters or lipopolysaccharide or by overexpression of constitutively active IK⌲␤. Functional characterization of RelA⅐RelB heterodimers in NIH3T3 murine embryonic fibroblasts revealed that they are not regulated by I〉 proteins and are located in both the cytoplasm and the nucleus. Taken together, our findings demonstrate that sequestration of RelA in transcriptionally inactive RelA⅐RelB complexes provides a molecular mechanism that may explain the repressive role of RelB on NF-〉-dependent gene expression.

T cell receptor (TCR) ligation induces increased diacylglycerol and Ca2؉ levels in T cells, and both secondary messengers are crucial for TCR-induced nuclear factor of activated T cells (NF-AT) and NF-B signaling pathways. One prominent calcium-dependent enzyme involved in the regulation of NF-AT and NF-B signaling pathways is the protein phosphatase calcineurin. However, in contrast to NF-AT, which is directly dephosphorylated by calcineurin, the molecular basis of the calcium-calcineurin dependence of the TCR-induced NF-B activity remains largely unknown. Here, we demonstrate that calcineurin regulates TCRinduced NF-B activity by controlling the formation of a protein complex composed of Carma1, Bcl10, and Malt1 (CBM complex). For instance, increased calcium levels induced by ionomycin or thapsigargin augmented the phorbol 12-myristate 13-acetate-induced formation of the CBM complex and activation of NF-B, whereas removal of calcium by the calcium chelator EGTA-acetoxymethyl ester (AM) attenuated both processes. Furthermore, inhibition of the calcium-dependent phosphatase calcineurin with the immunosuppressive agent cyclosporin A (CsA) or FK506 as well as siRNA-mediated knockdown of calcineurin A strongly affected the PMA ؉ ionomycin-or anti-CD3 ؉ CD28-induced CBM complex assembly. Mechanistically, the positive effect of calcineurin on the CBM complex formation seems to be linked to a dephosphorylation of Bcl10. For instance, Bcl10 was found to be hyperphosphorylated in Jurkat T cells upon treatment with CsA or EGTA-AM, and calcineurin dephosphorylated Bcl10 in vivo and in vitro. Furthermore, we show here that calcineurin A interacts with the CBM complex. In summary, the evidence provided here argues for a previously unanticipated role of calcineurin in CBM complex formation as a molecular basis of the inhibitory function of CsA or FK506 on TCR-induced NF-B activity.Upon engagement of the T cell receptor (TCR), 2 phospholipase C␥1 (PLC␥1) is induced, which in turn hydrolyzes phosphoinositol 4,5-bisphosphate, resulting in elevated levels of diacylglycerol and inositol trisphosphate. Diacylglycerol is crucial for the activation of members of the PKC family, with PKC⍜ being the key PKC family member required for the activation of AP1, NF-AT, and NF-B signaling pathways in T cells. The other product generated by phosphoinositol 4,5-bisphosphate hydrolysis, inositol trisphosphate, is an inducer of calcium influx into the cytoplasm of T cells by a process known as storeoperated calcium entry. After an initial inositol trisphosphateinduced depletion of the calcium stored in the endoplasmic reticulum, CRAC channels at the plasma membrane are opened in a STIM-dependent fashion, leading to a more sustained calcium influx (1). Calcium acts as a second messenger in the T cell necessary for the activation of several signaling molecules, including the protein kinases CaMKII and PKC␣ as well as the protein phosphatase calcineurin, which in turn are crucial for the activation of the transcription factors NF-AT, AP1, CREB1, an...

RelB is an unusual member of the Rel/NF-kB family of transcription factors which are involved in oncogenic processes. Due to a relaxed control by the IkBs, the cytosolic NF-kB inhibitors, RelB is constitutively expressed in the nuclei of lymphoid cells. We show here that RelB is inducibly degraded upon activation of T cells in a fashion similar to the IkBs. However, RelB degradation di ers from that of IkBs since it is not induced by TNFa but only by T cell receptor or TPA/ ionomycin stimulation. Moreover, RelB degradation occurs in three steps: (i) after stimulation RelB is rapidly phosphorylated at amino acids Thr84 and Ser552 followed by (ii) an N-terminal cut and, ®nally, (iii) the complete degradation in the proteasomes. Since mutation of the two phosphoacceptor sites to non-acceptor sites abolished RelB phosphorylation in vivo and led to the stabilization of the mutated RelB DM , site-speci®c phosphorylation appears to be a necessary prerequisite for RelB degradation. RelB is a crucial regulator of NFkB-dependent gene expression. Thus, the signal-induced degradation of RelB should be an important control mechanism of NF-kB activity. Oncogene (2001) 20, 8142 ± 8147.

Variants in the fat mass- and obesity-associated (FTO) gene are associated with obesity and body fat mass in genome-wide association studies. However, the mechanism by which FTO predisposes individuals to obesity is not clear so far. First mechanistic evidence was shown in Fto-negative mice. These mice are resistant to obesity due to enhanced energy expenditure, whereas the mass of brown adipose tissue remains unchanged. We hypothesize that FTO is involved in the induction of white adipose tissue browning, which leads to mitochondrial uncoupling and increases energy expenditure. Uncoupling protein 1 (Ucp-1) was significantly higher expressed in both gonadal and inguinal adipose depots of Fto(-/-) compared with Fto(+/+) littermates accompanied by the appearance of multivacuolar, Ucp-1-positive adipocytes in these tissues. By using lentiviral short hairpin RNA constructs, we established FTO-deficient human preadipocytes and adipocytes and analyzed key metabolic processes. FTO-deficient adipocytes showed an adipogenic differentiation rate comparable with control cells but exhibited a reduced de novo lipogenesis despite unchanged glucose uptake. In agreement with the mouse data, FTO-deficient adipocytes exhibited 4-fold higher expression of UCP-1 in mitochondria compared with control cells. The up-regulation of UCP-1 in FTO-deficient adipocytes resulted in enhanced mitochondrial uncoupling. We conclude that FTO deficiency leads to the induction of a brown adipocyte phenotype, thereby enhancing energy expenditure. Further understanding of the signaling pathway connecting FTO with UCP-1 expression might lead to new options for obesity and overweight treatment.

Chordomas are tumors that arise at vertebral bodies and the base of the skull. Although rare in incidence, they are deadly owing to slow growth and a lack of effective therapeutic options. In this study, we addressed the need for chordoma cell systems that can be used to identify therapeutic targets and empower testing of candidate pharmacologic drugs. Eight human chordoma cell lines that we established exhibited cytology, genomics, mRNA, and protein profiles that were characteristic of primary chordomas. Candidate responder profiles were identified through an immunohistochemical analysis of a chordoma tissue bank of 43 patients. Genomic, mRNA, and protein expression analyses confirmed that the new cell systems were highly representative of chordoma tissues. Notably, all cells exhibited a loss of CDKN2A and p16, resulting in universal activation of the CDK4/6 and Rb pathways. Therefore, we investigated the CDK4/6 pathway and responses to the CDK4/6-specific inhibitor palbociclib. In the newly validated system, palbociclib treatment efficiently inhibited tumor cell growth in vitro and a drug responder versus nonresponder molecular signature was defined on the basis of immunohistochemical expression of CDK4/6/pRb (S780). Overall, our work offers a valuable new tool for chordoma studies including the development of novel biomarkers and molecular targeting strategies. Cancer Res; 75(18); 3823-31. Ó2015 AACR.

The transcription factor NF-B plays a crucial role in the initiation of innate and adaptive immune responses, in inflammation and tumorigenesis (1-3). In its inactive state NF-B is bound to small cytoplasmic proteins, the IB proteins. Stimulation with a wide variety of agonists, for example pro-inflammatory cytokines like TNF-␣, 2 bacterial components like lipopolysaccharide or by antigen receptors, funnels in the activation of a multisubunit IB-kinase complex, which phosphorylates the IB proteins at two specific serine residues. This phosphorylation marks the IB proteins for proteasomal degradation setting NF-B free, which then translocates into the nucleus and supports the expression of various pro-inflammatory or anti-apoptotic gene products. The IB kinase (IKK) complex is composed of two catalytically active subunits, termed IKK␣ (IKK1) and IKK␤ (IKK2), as well as NEMO/IKK␥, a subunit with regulatory and adaptor functions (4, 5). Analysis of mice deficient for either IKK␣ or IKK␤ suggested that the IKK␤ subunit is the major IKK regulating the canonical NF-B pathway, and the IKK␣ subunit is crucial for a second, alternative NF-B pathway leading to the activation of RelB-p52 heterodimers (6, 7). In contrast to the canonical NF-B pathway, which depends on the presence of NEMO, the alternative pathway is NEMO-independent but requires the protein kinase NIK. However, recent data suggest that, instead of a clear assignment of IKK␣ and IKK␤ to the alternative and the canonical NF-B pathway, respectively, both kinases contribute to the activation of NF-B by the canonical pathway with only gradual differences (8,9). Although a precise model regarding the molecular mechanism underlying IKK activation is still missing, it became clear that various post-translational modifications at the different IKK subunits are involved in this process. Besides the phosphorylation of IKK␣ and IKK␤ at two serine residues in their T-loop, the ubiquitination and occasionally the phosphorylation at a specific serine residue of NEMO seem to be required (10 -12). This serine residue at position 85 of NEMO has been identified as a protein kinase C␣, and more recently as an ATM target-site crucial for the IKK activation induced by genotoxic stress (12, 13). In addition, overexpression of the TNF-␣-receptor I or the human T-cell lymphotrophic virus I Tax protein induces the IKK␤-mediated NEMO phosphorylation at several