The FKHR gene was first identified from its disruption by the t(2;13) chromosomal translocation seen in the pediatric tumor alveolar rhabdomyosarcoma. It encodes for a member of the forkhead family of transcription factors. Recently, a homolog of FKHR in the nematode Caenorhabditis elegans was identified called DAF-16, which is a downstream target of two Akt homologs in an insulin-related signaling pathway. We have examined the possible role of Akt in the regulation of FKHR. We find that FKHR can bind in vitro to the insulin-responsive sequence (IRS) in the insulinlike growth factor-binding protein 1 promoter and can activate transcription from a reporter plasmid containing multiple copies of the IRS. Expression of active but not inactive Akt can suppress FKHR-mediated transcriptional activation. Akt can phosphorylate FKHR in vitro on three phosphoacceptor sites, at least a subset of which can also be phosphorylated by Akt in vivo. Importantly, mutation of these three sites to alanine residues enhances the transcriptional activity of FKHR and renders it resistant to inhibition by Akt. Expression of an Akt-resistant mutant of FKHR causes apoptosis in 293T cells in a manner dependent on DNA binding. These results suggest that FKHR may be a direct nuclear regulatory target for Akt in both metabolic and cell survival pathways.

An imbalance in bone formation relative to bone resorption results in the net bone loss in osteoporosis and inflammatory bone diseases. While it is well known how bone resorption is stimulated, the molecular mechanisms that mediate impaired bone formation are poorly understood. Here we show that the time- and stage-specific inhibition of endogenous IκB kinase (IKK)/nuclear factor-kappa B (NF-κB) NF-κB in differentiated osteoblasts significantly increases trabecular bone mass and bone mineral density without affecting osteoclast activities in young mice. Moreover, the inhibition of IKK/NF-κB in differentiated osteoblasts maintains bone formation, thereby preventing osteoporotic bone loss induced by ovariectomy (OVX) in adult mice. The inhibition of IKK/NF-κB enhances the expression of Fra-1, an essential factor for bone matrix formation in vitro and in vivo. Taken together, our results suggest that targeting IKK/NF-κB may help to promote bone formation in the treatment of osteoporosis and other bone diseases.

Activation of the IκB kinase (IKK) complex by LPS induces phosphorylation and degradation of IκBα, leading to the nuclear translocation of NF-κB. Although it is essential for NF-κB activation, emerging evidence has indicated that the nuclear translocation of NF-κB is not sufficient to activate NF-κB-dependent transcription. Here, we reported that LPS induced the phosphorylation of the p65 trans-activation domain on serine 536 in monocytes/macrophages. Using mouse embryonic fibroblasts lacking either IKKα or IKKβ, we found that IKKβ played an essential role in LPS-induced p65 phosphorylation on serine 536, while IKKα was partially required for the p65 phosphorylation. The LPS-induced p65 phosphorylation on serine 536 was independent of the phosphatidylinositol 3′-kinase/Akt signaling pathway. Furthermore, we found that the phosphorylation on serine 536 increased the p65 transcription activity. In summary, our results demonstrate that IKKβ plays an essential role in the LPS-induced p65 phosphorylation on serine 536, which may represent a mechanism to regulate the NF-κB transcription activity by LPS.

NEMO (NF-B essential modifier)/IKK␥ (IB kinase-␥) is required for the activation of the IB kinase complex (IKK) by inflammatory stimuli such as tumor necrosis factor (TNF-␣).Here we show that TNF-␣ stimulates the ubiquitination of NEMO in a manner that does not appear to target it for degradation and that is impaired by mutations in the NEMO zinc finger. Mutations of the zinc finger are found in patients with hypohidrotic ectodermal dysplasia with immunodeficiency (HED-ID) and lead to the impairment of TNF-␣-stimulated IKK phosphorylation and activation. In addition, the ubiquitination of NEMO is mediated by c-IAP1, an inhibitor of apoptosis protein that is a component of the TNF receptor signaling complex. Thus, the ubiquitination of NEMO mediated by c-IAP1 likely plays an important role in the activation of IKK by TNF-␣. Also, defective NEMO ubiquitination may be responsible for the impaired cellular NF-B signaling found in patients with HED-ID.The NF-B/Rel family of transcription factors function in a wide range of biological activities including inflammation, immunity, and apoptosis, and their activities are regulated by their interactions with IB proteins (1-4). In unstimulated cells, NF-B is kept inactive in the cytoplasm by virtue of the masking of its nuclear localization sequence by bound IB protein. Exposure of cells to proinflammatory stimuli triggers the activation of a multisubunit IB kinase (IKK) 1 complex that phosphorylates IB proteins on two serine residues (5-7). Phosphorylation of IB proteins triggers their polyubiquitination and their subsequent recognition and degradation by the proteasome. Destruction of IB proteins liberates NF-B to enter the nucleus and activate gene expression (2,3,8).The predominant IKK complex found in cell lines contains two catalytic subunits, IKK␣ (or IKK1) and IKK␤ (or IKK2) (9 -13), and a regulatory subunit, NEMO (or IKK␥) (14 -17). IKK␣ and IKK␤ are serine/threonine protein kinases, whereas NEMO contains several protein interaction motifs but no apparent catalytic domains. Subunit reconstitution experiments in yeast and mammalian cells suggest that IKK is composed of a NEMO homodimer bound together with either an IKK␣/ IKK␤ heterodimer or an IKK␤ homodimer (18). Although structurally similar, IKK␣ and IKK␤ have distinct cellular functions. IKK␤ phosphorylates IB and is critical for IKK and NF-B activation in response to proinflammatory stimuli (19 -21). In contrast, IKK␣ phosphorylates the NF-B2/p100 precursor and stimulates its processing in a fashion that is independent of the IKK complex (22). The biochemical mechanisms underlying the activation of IKK in response to proinflammatory stimuli are not well understood. Although it is known that phosphorylation of Ser 179 and Ser 181 in the activation loop of IKK␤ is required and may be sufficient for IKK activation by proinflammatory stimuli (12, 23), the biochemical mechanisms regulating these phosphorylation events are not clear.NEMO was initially identified by complementation cloning in a rat cell line in whi...

No abstract

Active biofluid management is central to the realization of wearable bioanalytical platforms that are poised to autonomously provide frequent, real-time, and accurate measures of biomarkers in epidermally-retrievable biofluids (e.g., sweat). Accordingly, here, a programmable epidermal microfluidic valving system is devised, which is capable of biofluid sampling, routing, and compartmentalization for biomarker analysis. At its core, the system is a network of individually-addressable microheater-controlled thermo-responsive hydrogel valves, augmented with a pressure regulation mechanism to accommodate pressure built-up, when interfacing sweat glands. The active biofluid control achieved by this system is harnessed to create unprecedented wearable bioanalytical capabilities at both the sensor level (decoupling the confounding influence of flow rate variability on sensor response) and the system level (facilitating context-based sensor selection/protection). Through integration with a wireless flexible printed circuit board and seamless bilateral communication with consumer electronics (e.g., smartwatch), contextually-relevant (scheduled/on-demand) on-body biomarker data acquisition/display was achieved.

Phosphorylation can both positively and negatively regulate activity of the Raf kinases. Akt has been shown to phosphorylate and inhibit C-Raf activity. We have recently reported that Akt negatively regulates B-Raf kinase activation by phosphorylating multiple residues within its amino-terminal regulatory domain. Here we investigated the regulation of B-Raf by serum and glucocorticoid-inducible kinase, SGK, which shares close sequence identity with the catalytic domain of Akt but lacks the pleckstrin homology domain. We observed that SGK inhibits B-Raf activity. A comparison of substrate specificity between SGK and Akt indicates that SGK is a potent negative regulator of B-Raf. In contrast to Akt, SGK negatively regulates B-Raf kinase activity by phosphorylating only a single Akt consensus site, Ser 364 . Under similar experimental conditions, SGK displays a measurably stronger inhibitory effect on B-Raf kinase activity than Akt, whereas Akt exhibits a more inhibitory effect on the forkhead transcription factor, FKHR. The selective substrate specificity is correlated with an enhanced association between Akt or SGK and their preferred substrates, FKHR and B-Raf, respectively. These results indicate that B-Raf kinase activity is negatively regulated by Akt and SGK, suggesting that the cross-talk between the B-Raf and other signaling pathways can be mediated by both Akt and SGK.

The recognition of nucleic acids by the innate immune system during viral infection results in the production of type I interferons and the activation of antiviral immune responses. The RNA helicases RIG-I and MDA-5 recognize distinct types of cytosolic RNA species and signal through the mitochondrial protein MAVS to stimulate the phosphorylation and activation of the transcription factors IRF3 and IRF7, thereby inducing type I interferon expression. Alternatively, the activation of NF-κB leads to proinflammatory cytokine production. The function of MAVS is dependent on both its C-terminal transmembrane (TM) domain and N-terminal caspase recruitment domain (CARD). The TM domain mediates MAVS dimerization in response to viral RNA, allowing the CARD to bind to and activate the downstream effector TRAF3. Notably, dimerization of the MAVS CARD alone is sufficient to activate IRF3, IRF7, and NF-κB. However, TRAF3-deficient cells display only a partial reduction in interferon production in response to RNA virus infection and are not defective in NF-κB activation. Here we find that the related ubiquitin ligase TRAF5 is a downstream target of MAVS that mediates both IRF3 and NF-κB activation. The TM domain of MAVS allows it to dimerize and thereby associate with TRAF5 and induce its ubiquitination in a CARD-dependent manner. Also, NEMO is recruited to the dimerized MAVS CARD domain in a TRAF3 and TRAF5-dependent manner. Thus, our findings reveal a possible function for TRAF5 in mediating the activation of IRF3 and NF-κB downstream of MAVS through the recruitment of NEMO. TRAF5 may be a key molecule in the innate response against viral infection.