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The activation of transcription by light in the fungus Neurospora crassa requires the White Collar Complex (WCC), a photoreceptor and transcription factor complex. After light reception two WCCs interact and bind the promoters of light- regulated genes to activate transcription. This process is regulated by VVD, a small photoreceptor that disrupts the interaction between WCCs and leads to a reduction in transcription after long exposures to light. The N. crassa RCO-1/RCM-1 repressor complex is the homolog of the Tup1-Ssn6 repressor complex in yeast, and its absence modifies photoadaptation. We show that the absence of the RCO-1/RCM-1 repressor complex leads to several alterations in transcription that are gene-specific: an increase in the accumulation of mRNAs in the dark, a repression of transcription, and a derepression of transcription after long exposures to light. The absence of the RCO-1/RCM-1 repressor complex leads to lower VVD levels that are available for the regulation of the activity of the WCC. The reduction in the amount of VVD results in increased WCC binding to the promoters of light-regulated genes in the dark and after long exposures to light, leading to the modification of photoadaptation that has been observed in rco-1 and rcm-1 mutants. Our results show that the photoadaptation phenotype of mutants in the RCO-1/RCM-1 repressor complex is, at least in part, an indirect consequence of the reduction of vvd transcription, and the resulting modification in the regulation of transcription by the WCC

Formation of signaling protein complexes is crucial for proper signal transduction. Scaffold proteins in MAP kinase pathways are thought to facilitate complex assembly, thereby promoting efficient and specific signaling. To elucidate the assembly mechanism of scaffold complexes in mammals, we attempted to rationally rewire JIP1-dependent JNK MAP kinase pathway via alternative assembly of JIP1 complex. When JIP1-JNK docking interaction in the complex was replaced with heterologous protein interaction domains, such as PDZ domains and JNK-binding domains, a functional scaffold complex was reconstituted, and JNK signaling was rescued. Reassembly of JIP1 complex using heterologous protein interactions was sufficient for restoring of JNK MAP kinase pathway to induce signaling responses, including JNK activation and cell death. These results suggest a simple yet modular mechanism for JIP1 scaffold assembly in mammals.

The subcellular localization of the epidermal growth factor receptor (EGFR) in polarized epithelial cells profoundly affects the activity of the intracellular signaling pathways activated after EGF ligand binding. Therefore, changes in EGFR localization and signaling are implicated in various human diseases, including different types of cancer. We have performed the first in vivo EGFR localization screen in an animal model by observing the expression of the EGFR ortholog LET-23 in the vulval epithelium of live C. elegans larvae. After systematically testing all genes known to produce an aberrant vulval phenotype, we have identified 81 genes regulating various aspects of EGFR localization and expression. In particular, we have found that ERM-1, the sole C. elegans Ezrin/Radixin/Moesin homolog, regulates EGFR localization and signaling in the vulval cells. ERM-1 interacts with the EGFR at the basolateral plasma membrane in a complex distinct from the previously identified LIN-2/LIN-7/LIN-10 receptor localization complex. We propose that ERM-1 binds to and sequesters basolateral LET-23 EGFR in an actin-rich inactive membrane compartment to restrict receptor mobility and signaling. In this manner, ERM-1 prevents the immediate activation of the entire pool of LET-23 EGFR and permits the generation of a long-lasting inductive signal. The regulation of receptor localization thus serves to fine-tune the temporal activation of intracellular signaling pathways., PLoS Genetics, 10 (5), ISSN:1553-7390, ISSN:1553-7404

Celastrol, a tripterine derived from the traditional Chinese medicine plant Tripterygium wilfordii Hook F. (“Thunder of God Vine”), has been reported to have multiple effects, such as anti-inflammation, suppression of tumor angiogenesis, inhibition of tumor growth, induction of apoptosis and protection of cells against human neurodegenerative diseases. However, the mechanisms that underlie these functions are not well defined. In this study, we reported for the first time that Celastrol could induce HIF-1α protein accumulation in multiple cancer cell lines in an oxygen-independent manner and that the enhanced HIF-1α protein entered the nucleus and promoted the transcription of the HIF-1 target genes VEGF and Glut-1. Celastrol did not influence HIF-1α transcription. Instead, Celastrol induced the accumulation of the HIF-1α protein by inducing ROS and activating Akt/p70S6K signaling to promote HIF-1α translation. In addition, we found that the activation of Akt by Celastrol was transient. With increased exposure time, inhibition of Hsp90 chaperone function by Celastrol led to the subsequent depletion of the Akt protein and thus to the suppression of Akt activity. Moreover, in HepG2 cells, the accumulation of HIF-1α increased the expression of BNIP3, which induced autophagy. However, HIF-1α and BNIP3 did not influence the cytotoxicity of Celastrol because the main mechanism by which Celastrol kills cancer cells is through stimulating ROS-mediated JNK activation and inducing apoptosis. Furthermore, our data showed that the dose required for Celastrol to induce HIF-1α protein accumulation and enhance HIF-1α transcriptional activation was below its cytotoxic threshold. A cytotoxic dose of Celastrol for cancer cells did not display cytotoxicity in LO2 normal human liver cells, which indicated that the novel functions of Celastrol in regulating HIF-1 signaling and inducing autophagy might be used in new applications, such as in anti-inflammation and protection of cells against human neurodegenerative diseases. Future studies regarding these applications are required.

Protease-activated receptor-2 is widely expressed in mammalian epithelial, immune and neural tissues. Cleavage of PAR2 by serine proteases leads to self-activation of the receptor by the tethered ligand SLIGRL. The contribution of other classes of proteases to PAR activation has not been studied in detail. Cathepsin S is a widely expressed cysteine protease that is upregulated in inflammatory conditions. It has been suggested that cathepsin S activates PAR2. However, cathepsin S activation of PAR2 has not been demonstrated directly nor has the potential mechanism of activation been identified. We show that cathepsin S cleaves near the N-terminus of PAR2 to expose a novel tethered ligand, KVDGTS. The hexapeptide KVDGTS generates downstream signaling events specific to PAR2 but is weaker than SLIGRL. Mutation of the cathepsin S cleavage site prevents receptor activation by the protease while KVDGTS retains activity. In conclusion, the range of actions previously ascribed to cysteine cathepsins in general, and cathepsin S in particular, should be expanded to include molecular signaling. Such signaling may link together observations that had been attributed previously to PAR2 or cathepsin S individually. These interactions may contribute to inflammation.

Smad Anchor for Receptor Activation (SARA) has been reported as a critical role in TGF-β signal transduction by recruiting non-activated Smad2/3 to the TGF-β receptor and ensuring appropriate subcellular localization of the activated receptor-bound complex. However, controversies still exist in previous reports. In this study, we describe the expression of two SARA isoforms, SARA1 and SARA2, in mice and report the generation and characterization of SARA mutant mice with FYVE domain deletion. SARA mutant mice developed normally and showed no gross abnormalities. Further examination showed that the TGF-β signaling pathway was indeed altered in SARA mutant mice, with the downregulation of Smad2 protein expression. The decreasing expression of Smad2 was caused by enhancing Smurf2-mediated proteasome degradation pathway. However, the internalization of TGF-β receptors into the early endosome was not affected in SARA mutant mouse embryonic fibroblasts (MEFs). Moreover, the downregulation of Smad2 in SARA mutant MEFs was not sufficient to disrupt the diverse cellular biological functions of TGF-β signaling, including growth inhibition, apoptosis, senescence, and the epithelial-to-mesenchymal transition. Our results indicate that SARA is not involved in the activation process of TGF-β signal transduction. Using a two-stage skin chemical carcinogenesis assay, we found that the loss of SARA promoted skin tumor formation and malignant progression. Our data suggest a protective role of SARA in skin carcinogenesis.

International audience; The pathogenesis of complex diseases, such as type 1 diabetes (T1D), derives from interactions between host genetics and environmental factors. Previous studies have suggested that viral infection plays a significant role in initiation of T1D in genetically predisposed individuals. T1D susceptibility loci may therefore be enriched in previously uncharacterized genes functioning in antiviral defense pathways. To identify genes involved in antiviral immunity, we performed an image-based high-throughput genetic screen using short hairpin RNAs (shRNAs) against 161 genes within T1D susceptibility loci. RAW 264.7 cells transduced with shRNAs were infected with GFP-expressing herpes simplex virus type 1 (HSV-1) and fluorescent microscopy was performed to assess the viral infectivity by fluorescence reporter activity. Of the 14 candidates identified with high confidence, two candidates were selected for further investigation, Il27 and Tagap. Administration of recombinant IL-27 during viral infection was found to act synergistically with interferon gamma (IFN-γ) to activate expression of type I IFNs and proinflammatory cytokines, and to enhance the activities of interferon regulatory factor 3 (IRF3). Consistent with a role in antiviral immunity, Tagap-deficient macrophages demonstrated increased viral replication, reduced expression of proinflammatory chemokines and cytokines, and decreased production of IFN-β. Taken together, our unbiased loss-of-function genetic screen identifies genes that play a role in host antiviral immunity and delineates roles for IL-27 and Tagap in the production of antiviral cytokines.

It has been proposed that differential activation kinetics allows cells to use a common set of signaling pathways to specify distinct cellular outcomes. For example, nerve growth factor (NGF) and epidermal growth factor (EGF) induce different activation kinetics of the Raf/MEK/ERK signaling pathway and result in differentiation and proliferation, respectively. However, a direct and quantitative linkage between the temporal profile of Raf/MEK/ERK activation and the cellular outputs has not been established due to a lack of means to precisely perturb its signaling kinetics. Here, we construct a light-gated protein-protein interaction system to regulate the activation pattern of the Raf/MEK/ERK signaling pathway. Light-induced activation of the Raf/MEK/ERK cascade leads to significant neurite outgrowth in rat PC12 pheochromocytoma cell lines in the absence of growth factors. Compared with NGF stimulation, light stimulation induces longer but fewer neurites. Intermittent on/off illumination reveals that cells achieve maximum neurite outgrowth if the off-time duration per cycle is shorter than 45 min. Overall, light-mediated kinetic control enables precise dissection of the temporal dimension within the intracellular signal transduction network.

Background Magnetic nanoparticles (NPs) are of particular interest in biomedical research, and have been exploited for molecular separation, gene/drug delivery, magnetic resonance imaging, and hyperthermic cancer therapy. In the case of cultured cells, magnetic manipulation of NPs provides the means for studying processes induced by mechanotransduction or by local clustering of targeted macromolecules, e.g. cell surface receptors. The latter are normally activated by binding of their natural ligands mediating key signaling pathways such as those associated with the epidermal growth factor (EGFR). However, it has been reported that EGFR may be dimerized and activated even in the absence of ligands. The present study assessed whether receptor clustering induced by physical means alone suffices for activating EGFR in quiescent cells. Methodology/Principal Findings The EGFR on A431 cells was specifically targeted by superparamagnetic iron oxide NPs (SPIONs) carrying either a ligand-blocking monoclonal anti-EGFR antibody or a streptavidin molecule for targeting a chimeric EGFR incorporating a biotinylated amino-terminal acyl carrier peptide moiety. Application of a magnetic field led to SPION magnetization and clustering, resulting in activation of the EGFR, a process manifested by auto and transphosphorylation and downstream signaling. The magnetically-induced early signaling events were similar to those inherent to the ligand dependent EGFR pathways. Magnetization studies indicated that the NPs exerted magnetic dipolar forces in the sub-piconewton range with clustering dependent on Brownian motion of the receptor-SPION complex and magnetic field strength. Conclusions/Significance We demonstrate that EGFR on the cell surface that have their ligand binding-pocket blocked by an antibody are still capable of transphosphorylation and initiation of signaling cascades if they are clustered by SPIONs either attached locally or targeted to another site of the receptor ectodomain. The results suggest that activation of growth factor receptors may be triggered by ligand-independent molecular crowding resulting from overexpression and/or sequestration in membrane microdomains.

Background Insulin-like growth factor binding proteins (IGFBPs) are six related secreted proteins that share IGF-dependent and -independent functions. If the former functions begin to be well described, the latter are somewhat more difficult to investigate and to characterize. At the cellular level, IGFBPs were shown to modulate numerous processes including cell growth, differentiation and apoptosis. However, the molecular mechanisms implicated remain largely unknown. We previously demonstrated that IGFBP-3, but not IGFBP-1 or IGFBP-5, increase intracellular calcium concentration in MCF-7 cells (Ricort J-M et al. (2002) FEBS lett 527: 293–297). Methodology/Principal Findings We perform a global analysis in which we studied, by two different approaches, the binding of each IGFBP isoform (i.e., IGFBP-1 to -6) to the surface of two different cellular models, MCF-7 breast adenocarcinoma cells and C2 myoblast proliferative cells, as well as the IGFBP-induced increase of intracellular calcium concentration. Using both confocal fluorescence microscopy and flow cytometry analysis, we showed that all IGFBPs bind to MCF-7 cell surface. By contrast, only four IGFBPs can bind to C2 cell surface since neither IGFBP-2 nor IGFBP-4 were detected. Among the six IGFBPs tested, only IGFBP-1 did not increased intracellular calcium concentration whatever the cellular model studied. By contrast, IGFBP-2, -3, -4 and -6, in MCF-7 cells, and IGFBP-3, -5 and -6, in C2 proliferative cells, induce a rapid and transient increase in intracellular free calcium concentration. Moreover, IGFBP-2 and -3 (in MCF-7 cells) and IGFBP-5 (in C2 cells) increase intracellular free calcium concentration by a pertussis toxin sensitive signaling pathway. Conclusions Our results demonstrate that IGFBPs are able to bind to cell surface and increase intracellular calcium concentration. By characterizing the IGFBPs-induced cell responses and intracellular couplings, we highlight the cellular specificity and complexity of the IGF-independent actions of these IGF binding proteins.

Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation.

BACKGROUND: The phosphatase PTEN represents an important physiological inhibitor of phosphatidylinositol-3 kinase (PI3-K)/protein kinase B (Akt) signalling, however, the functional role of PTEN in the initial phase of angioplasty-induced vascular injury remains elusive. In the present study we sought to determine PTEN's effect on vascular smooth muscle cell (VSMC) apoptosis following acute injury in vivo and in vitro. METHODS AND RESULTS: Immunohistochemistry indicated a faint basal expression and equal distribution of PTEN in uninjured rat carotid arteries. 12 h following balloon-injury, PTEN expression was strongly increased in apoptotic (TUNEL+) VSMC. In vitro, stimulation with serum or different growth factors or subjecting VSMC to cyclic stretch had no effect on PTEN expression, whereas stimulation with H2O2 robustly increased PTEN expression in a time- and dose-dependent manner. To evaluate the functional role of PTEN expression, human VSMC were transduced with WT-PTEN. Overexpression of PTEN increased the number of apoptotic VSMC (19.8%±4.4 vs. 5.6%±2.3; P

Background Skin cancer is the most common cancer throughout the world. The epithelial-mesenchymal transition (EMT) and the acquisition of cancer stem cells (CSCs)-like properties emerge as critical steps in the metastasis of human skin cancers. Caffeic acid (CaA) exerts anticarcinogenic effects. However, the effects of CaA on the migratory capability and on the CSCs-like properties of skin cancer cells, and the molecular mechanisms underlying it are not fully understood. Methods Malignant HaCaT cells were treated by CaA. Transwell assay was performed to determine that CaA attenuated the migratory capability; Spheroid formation assay was performed to confirm that CaA decreased the CSCs-like phenotype; Treated malignant HaCaT cells were molecularly characterized by RT-PCR, Western blots, Southwestern blot, and immunoprecipitation. Results In CaA-treated malignant human keratinocyte (malignant HaCaT cells), inhibition of the migratory capability and CSCs-like phenotype were observed. CaA up-regulated the phosphorylation of p38, and down-regulated the activation of nuclear factor κB (NF-κB)/snail signal pathway. Indeed, p38 decreased the DNA-binding activity of NF-κB to the promoter of snail gene, which resulted in the transcriptional inactivation of snail. Blockage of p38 attenuated the CaA-induced inhibition of migratory capability and CSCs-like phenotype in malignant HaCaT cells. Conclusions CaA attenuates the migratory capability and CSCs-like Properties of malignant human keratinocyte, in which, p38-mediated down-regulation of NF-κB/snail signal pathway is involved.

Purinergic receptor expression and involvement in steroidogenesis were examined in NCI-H295R (H295R), a human adrenal cortex cell line which expresses all the key enzymes necessary for steroidogenesis. mRNA/protein for multiple P1 (A(2A) and A(2B)), P2X (P2X₅ and P2X₇), and P2Y (P2Y₁, P2Y₂, P2Y₆, P2Y₁₂, P2Y₁₃, and P2Y₁₄) purinergic receptors were detected in H295R. 2MeS-ATP (10-1000 µM), a P2Y₁ agonist, induced glucocorticoid (GC) secretion in a dose-dependent manner, while other extracellular purine/pyrimidine agonists (1-1000 µM) had no distinct effect on GC secretion. Extracellular purines, even non-steroidogenic ones, induced Ca²⁺-mobilization in the cells, independently of the extracellular Ca²⁺ concentration. Increases in intracellular Ca²⁺ concentration induced by extracellular purine agonists were transient, except when induced by ATP or 2MeS-ATP. Angiotensin II (AngII: 100 nM) and dibutyryl-cyclic AMP (db-cAMP: 500 µM) induced both GC secretion and Ca²⁺-mobilization in the presence of extracellular Ca²⁺ (1.2 mM). GC secretion by AngII was reduced by nifedipine (10-100 µM); whereas the Ca²⁺ channel blocker did not inhibit GC secretion by 2MeS-ATP. Thapsigargin followed by extracellular Ca²⁺ exposure induced Ca²⁺-influx in H295R, and the cells expressed mRNA/protein of the component molecules for store-operated calcium entry (SOCE): transient receptor C (TRPC) channels, calcium release-activated calcium channel protein 1 (Orai-1), and the stromal interaction molecule 1 (STIM1). In P2Y₁-knockdown, 2MeS-ATP-induced GC secretion was significantly inhibited. These results suggest that H295R expresses a functional P2Y₁ purinergic receptor for intracellular Ca²⁺-mobilization, and that P2Y₁ is linked to SOCE-activation, leading to Ca²⁺-influx which might be necessary for glucocorticoid secretion.

B cells are an integral component in mounting humoral immune responses and they are also crucial in programming T cell mediated immunity. Usually, B cell activation is initiated by recognition of antigen through B cell receptor (BCR), followed by its processing and presentation to T cells. But very little is known about BCR independent activation of B cells. Now, there is an increasing body of evidence indicating the combinatorial effect of innate and adaptive immune components in modulating the functions of B cells. In this study, we demonstrate the activation of resting B cells (RB) by simultaneous involvement of Toll like Receptor-2 (TLR-2) and costimulatory molecule, CD86. Interestingly, these B cells exhibited significant level of activation and proliferation. Furthermore, this process of activation leads to the differentiation of RB cells, preferably into marginal zone precursors (CD19(+)IgD(hi)IgM(hi)CD21/35(hi)CD23(hi)) in a shorter time window and showed increased secretion of IgG isotype. These RB cells also showed enhanced antigen uptake capacity. These observations were also substantiated by microarray gene expression results, which strengthen the notion that combinatorial signaling through innate and adaptive immune components enhances B cell mediated immune response. Thus, the present study elucidates a novel BCR independent B cell activation mechanism that links TLR-2 and CD86. Hence signaling of TLRs in conjunction with costimulatory molecules will substantially help in bolstering humoral immune response, which can be extrapolated to formulate vaccination strategies for diseases involving B cell-mediated immunity.

The major effects of cannabinoids and endocannabinoids are mediated via two G protein-coupled receptors, CB1 and CB2, elucidation of the mechanism and structural determinants of the CB2 receptor coupling with G proteins will have a significant impact on drug discovery. In the present study, we systematically investigated the role of the intracellular loops in the interaction of the CB2 receptor with G proteins using chimeric receptors alongside the characterization of cAMP accumulation and ERK1/2 phosphorylation. We provided evidence that ICL2 was significantly involved in G protein coupling in coordination with the C-terminal end. Moreover, a single alanine substitution of the Pro-139 in the CB2 receptor that corresponds to Leu-222 in the CB1 receptor resulted in a moderate impairment in the inhibition of cAMP accumulation, whereas mutants P139F, P139M and P139L were able to couple to the Gs protein in a CRE-driven luciferase assay. With the ERK activation experiments, we further found that P139L has the ability to activate ERK through both Gi- and Gs-mediated pathways. Our findings defined an essential role of the second intracellular loop of the CB2 receptor in coordination with the C-terminal tail in G protein coupling and receptor activation.

CD81 (TAPA-1) is a ubiquitously expressed tetraspanin protein identified as a component of the B lymphocyte receptor (BCR) and as a receptor for the Hepatitis C Virus. In an effort to identify trans-membrane proteins that interact with the T-cell antigen receptor (TCR), we performed a membrane yeast two hybrid screen and identified CD81 as an interactor of the CD3delta subunit of the TCR. We found that in the absence of CD81, in thymocytes from knockout mice, TCR engagement resulted in stronger signals. These results were recapitulated in T cell lines that express low levels of CD81 through shRNA mediated silencing. Increased signaling did not result from alterations in the levels of TCR on the surface of T lymphocytes. Although CD81 is not essential for normal T lymphocyte development, it plays an important role in regulating TCR and possibly pre-TCR signal transduction by controlling the strength of signaling. CD81 dependent alterations in thymocyte signaling are evident in increased CD5 expression on CD81 deficient double positive (DP) thymocytes. We conclude that CD81 interacts with the T cell receptor to suppress signaling. © 2012 Cevik et al.

Malignant transformation results in abnormal cell cycle regulation and uncontrolled growth in head and neck squamous cell carcinoma (HNSCC) and other cancers. S100A8/A9 (calprotectin) is a calcium-binding heterodimeric protein complex implicated in cell cycle regulation, but the specific mechanism and role in cell cycle control and carcinoma growth are not well understood. In HNSCC, S100A8/A9 is downregulated at both mRNA and protein levels. We now report that downregulation of S100A8/A9 correlates strongly with a loss of cell cycle control and increased growth of carcinoma cells. To show its role in carcinogenesis in an in vitro model, S100A8/A9 was stably expressed in an S100A8/A9-negative human carcinoma cell line (KB cells, HeLa-like). S100A8/A9 expression increases PP2A phosphatase activity and p-Chk1 (Ser345) phosphorylation, which appears to signal inhibitory phosphorylation of mitotic p-Cdc25C (Ser216) and p-Cdc2 (Thr14/Tyr15) to inactivate the G2/M Cdc2/cyclin B1 complex. Cyclin B1 expression then downregulates and the cell cycle arrests at the G2/M checkpoint, reducing cell division. As expected, S100A8/A9-expressing cells show both decreased anchorage-dependent and -independent growth and mitotic progression. Using shRNA, silencing of S100A8/A9 expression in the TR146 human HNSCC cell line increases growth and survival and reduces Cdc2 inhibitory phosphorylation at Thr14/Tyr15. The level of S100A8/A9 endogenous expression correlates strongly with the reduced p-Cdc2 (Thr14/Tyr14) level in HNSCC cell lines, SCC-58, OSCC-3 and UMSCC-17B. S100A8/A9-mediated control of the G2/M cell cycle checkpoint is, therefore, a likely suppressive mechanism in human squamous cell carcinomas and may suggest new therapeutic approaches.

It is well established that EGFR signals from both the plasma membrane (PM) and endosome (EN). However, very little is known about whether and how the EGFR signals at the PM and EN to differentially regulate various signaling pathways and the physiological outcomes. In this communication, we established a system that allowed the specific activations of EGFR at different cell locations: PM and EN. PM activation of EGFR is achieved by activation of endocytosis-deficient mutant EGFR1010LL/AA stably expressed in CHO cells (CHO-LL/AA cell). EN activation of EGFR is achieved by activating the wild type EGFR stably expressed in CHO cells (CHO-EGFR cell) after its internalization into EN with a previously reported protocol. We showed that both EGFR activations at PM and EN activated ERK to a similar level, but differentially stimulated transcriptional factors c-jun and c-fos. We further showed that EGFR activations at PM and EN resulted in differential spatio-temporal dynamics of phosphorylated ERK which caused the differential activation of two downstream substrates ELK1 and RSK. Finally we showed that EGFR signaling from PM and EN led to different physiological outcomes. CHO-LL/AA cells that only generate PM EGFR signals have a larger cell size and slower proliferation rate than CHO-EGFR cells. We conclude that location-specific EGFR activation differentially regulates cell functions through a spatio-temporal interplay of ERK activation.

The respiratory epithelium is subject to continuous environmental stress and its responses to injury or infection are largely mediated by transactivation of the epidermal growth factor receptor (EGFR) and downstream signaling cascades. Based on previous studies indicating involvement of ATP-dependent activation of the NADPH oxidase homolog DUOX1 in epithelial wound responses, the present studies were performed to elucidate the mechanisms by which DUOX1-derived H(2)O(2) participates in ATP-dependent redox signaling and EGFR transactivation. ATP-mediated EGFR transactivation in airway epithelial cells was found to involve purinergic P2Y(2) receptor stimulation, and both ligand-dependent mechanisms as well as ligand-independent EGFR activation by the non-receptor tyrosine kinase Src. Activation of Src was also essential for ATP-dependent activation of the sheddase ADAM17, which is responsible for liberation and activation of EGFR ligands. Activation of P2Y(2)R results in recruitment of Src and DUOX1 into a signaling complex, and transient siRNA silencing or stable shRNA transfection established a critical role for DUOX1 in ATP-dependent activation of Src, ADAM17, EGFR, and downstream wound responses. Using thiol-specific biotin labeling strategies, we determined that ATP-dependent EGFR transactivation was associated with DUOX1-dependent oxidation of cysteine residues within Src as well as ADAM17. In aggregate, our findings demonstrate that DUOX1 plays a central role in overall epithelial defense responses to infection or injury, by mediating oxidative activation of Src and ADAM17 in response to ATP-dependent P2Y(2)R activation as a proximal step in EGFR transactivation and downstream signaling.

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) ligand family that exerts its apoptotic activity in human cells by binding to two transmembrane receptors, TRAILR1 and TRAILR2. In cells co-expressing both receptors the particular contribution of either protein to the overall cellular response is not well defined. Here we have investigated whether differences in the signaling capacities of TRAILR1 and TRAILR2 can be attributed to certain functional molecular subdomains. We generated and characterized various chimeric receptors comprising TRAIL receptor domains fused with parts from other members of the TNF death receptor family. This allowed us to compare the contribution of particular domains of the two TRAIL receptors to the overall apoptotic response and to identify elements that regulate apoptotic signaling. Our results show that the TRAIL receptor death domains are weak apoptosis inducers compared to those of CD95/Fas, because TRAILR-derived constructs containing the CD95/Fas death domain possessed strongly enhanced apoptotic capabilities. Importantly, major differences in the signaling strengths of the two TRAIL receptors were linked to their transmembrane domains in combination with the adjacent extracellular stalk regions. This was evident from receptor chimeras comprising the extracellular part of TNFR1 and the intracellular signaling part of CD95/Fas. Both receptor chimeras showed comparable ligand binding affinities and internalization kinetics. However, the respective TRAILR2-derived molecule more efficiently induced apoptosis. It also activated caspase-8 and caspase-3 more strongly and more quickly, albeit being expressed at lower levels. These results suggest that the transmembrane domains together with their adjacent stalk regions can play a major role in control of death receptor activation thereby contributing to cell type specific differences in TRAILR1 and TRAILR2 signaling.

Signaling by mTOR is a well-recognized component of the pathway through which mechanical signals regulate protein synthesis and muscle mass. However, the mechanisms involved in the mechanical regulation of mTOR signaling have not been defined. Nevertheless, recent studies suggest that a mechanically-induced increase in phosphatidic acid (PA) may be involved. There is also evidence which suggests that mechanical stimuli, and PA, utilize ERK to induce mTOR signaling. Hence, we reasoned that a mechanically-induced increase in PA might promote mTOR signaling via an ERK-dependent mechanism. To test this, we subjected mouse skeletal muscles to mechanical stimulation in the presence or absence of a MEK/ERK inhibitor, and then measured several commonly used markers of mTOR signaling. Transgenic mice expressing a rapamycin-resistant mutant of mTOR were also used to confirm the validity of these markers. The results demonstrated that mechanically-induced increases in p70(s6k) T389 and 4E-BP1 S64 phosphorylation, and unexpectedly, a loss in total 4E-BP1, were fully mTOR-dependent signaling events. Furthermore, we determined that mechanical stimulation induced these mTOR-dependent events, and protein synthesis, through an ERK-independent mechanism. Similar to mechanical stimulation, exogenous PA also induced mTOR-dependent signaling via an ERK-independent mechanism. Moreover, PA was able to directly activate mTOR signaling in vitro. Combined, these results demonstrate that mechanical stimulation induces mTOR signaling, and protein synthesis, via an ERK-independent mechanism that potentially involves a direct interaction of PA with mTOR. Furthermore, it appears that a decrease in total 4E-BP1 may be part of the mTOR-dependent mechanism through which mechanical stimuli activate protein synthesis.

Escape from the host immune system is essential for intracellular pathogens. The adenoviral protein E3-14.7K (14.7K) is known as a general inhibitor of tumor necrosis factor (TNF)-induced apoptosis. It efficiently blocks TNF-receptor 1 (TNFR1) internalization but the underlying molecular mechanism still remains elusive. Direct interaction of 14.7K and/or associated proteins with the TNFR1 complex has been discussed although to date not proven. In our study, we provide for the first time evidence for recruitment of 14.7K and the 14.7K interacting protein optineurin to TNFR1. Various functions have been implicated for optineurin such as regulation of receptor endocytosis, vesicle trafficking, regulation of the nuclear factor kappa B (NF-kappa B) pathway and antiviral signaling. We therefore hypothesized that binding of optineurin to 14.7K and recruitment of both proteins to the TNFR1 complex is essential for protection against TNF-induced cytotoxic effects. To precisely dissect the individual role of 14.7K and optineurin, we generated and characterized a 14.7K mutant that does not confer TNF-resistance but is still able to interact with optineurin. In H1299 and KB cells expressing 14.7K wild-type protein, neither decrease in cell viability nor cleavage of caspases was observed upon stimulation with TNF. In sharp contrast, cells expressing the non-protective mutant of 14.7K displayed reduced viability and cleavage of initiator and effector caspases upon TNF treatment, indicating ongoing apoptotic cell death. Knockdown of optineurin in 14.7K expressing cells did not alter the protective effect as measured by cell viability and caspase activation. Taken together, we conclude that optineurin despite its substantial role in vesicular trafficking, endocytosis of cell surface receptors and recruitment to the TNFR1 complex is dispensable for the 14.7K-mediated protection against TNF-induced apoptosis.

Three distinct classes of nuclear receptors, EcR, E75, and HR3, are key regulators in the ecdysone-inducible gene activation cascade in insects. The transcription of these genes is induced by ecdysone (20E) differently, although the detailed mechanisms underlying their responses to 20E are largely unknown. We identified ecdysone response elements (EcREs) present in the promoters of genes coding BmEcR-B1, BmE75-A, and BHR3-B isoforms from Bombyx mori employing luciferase reporter assays in an ecdysteroid-responsive cultured cell line, NIAS-Bm-aff3 (aff3). The EcRE of BmEcR-B1 at -2800 comprises of two adjacent elements separated by 5 bp, E1 (15 bp) and E2 (21 bp), both of which are required for the 20E response. Further analysis using electrophoretic mobility shift assays showed that E1 binds to the EcR/USP heterodimer and that E2 may bind to the E-box (CACGTG) binding factor such as bHLH protein. The unique E1+E2-type EcRE is also detected in the promoter upstream regions of EcR-B1 from seven lepidopteran species studied. In contrast, both a 20 bp EcRE identified in the promoter of BmE75-A and a 18 bp EcRE identified in the BHR3-B promoter, contained only E1-type EcR/USP binding element but the E2 type element was not in the promoter regions of these genes. The combination of presence of the E2 element or other cis-regulatory elements in promoter regions explains the different 20E response of each class of nuclear receptor genes. Furthermore, the E1+E2 structure for EcR-B1 can be involved in a possible cross-talk between ecdysteroid and other regulatory pathways.

NF-E2 related factor-2 (Nrf2) promotes the transcription of many cytoprotective genes and is a major drug target for prevention of cancer and other diseases. Indeed, the cancer-preventive activities of several well-known chemical agents were shown to depend on Nrf2 activation. It is well known that chemopreventive Nrf2 activators stabilize Nrf2 by blocking its ubiquitination, but previous studies have indicated that this process occurs exclusively in the cytoplasm. Kelch-like ECH-associated protein 1 (Keap1) binds to Nrf2 and orchestrates Nrf2 ubiquitination, and it has been a widely-held view that inhibition of Nrf2 ubiquitination by chemopreventive agents results from the dissociation of Nrf2 from its repressor Keap1. Here, we show that while the activation of Nrf2 by prototypical chemical activators, including 5,6-dihydrocyclopenta-1,2-dithiole-3-thione (CPDT) and sulforaphane (SF), results solely from inhibition of its ubiquitination, such inhibition occurs predominantly in the nucleus. Moreover, the Nrf2 activators promote Nrf2 association with Keap1, rather than disassociation, which appears to result from inhibition of Nrf2 phosphorylation at Ser40. Available evidence suggests the Nrf2 activators may block Nrf2 ubiquitination by altering Keap1 conformation via reaction with the thiols of specific Keap1 cysteines. We further show that while the inhibitory effects of CPDT and SF on Nrf2 ubiquitination depend entirely on Keap1, Nrf2 is also degraded by a Keap1-independent mechanism. These findings provide significant new insight about Nrf2 activation and suggest that exogenous chemical activators of Nrf2 enter the nucleus to exert most of their inhibitory impact on Nrf2 ubiquitination and degradation.

Upon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling receptor signal fidelity. Here we show a critical role for membrane raft (MR) microdomains in creation of discrete signaling platforms essential for Epo-R signaling. Treatment of UT7 cells with Epo induced MR assembly and coalescence. Confocal microscopy showed that raft aggregates significantly increased after Epo stimulation (mean, 4.3 +/- 1.4(SE) vs. 25.6 +/- 3.2 aggregates/cell; p 3-fold increase in cluster size (p

Prediction of therapeutic response and cancer patient survival can be improved by the identification of molecular markers including tumor Akt status. A direct correlation between NOS2 expression and elevated Akt phosphorylation status has been observed in breast tumors. Tissue inhibitor matrix metalloproteinase-1 (TIMP-1) has been proposed to exert oncogenic properties through CD63 cell surface receptor pathway initiation of pro-survival PI3k/Akt signaling. We employed immunohistochemistry to examine the influence of TIMP-1 on the functional relationship between NOS2 and phosphorylated Akt in breast tumors and found that NOS2-associated Akt phosphorylation was significantly increased in tumors expressing high TIMP-1, indicating that TIMP-1 may further enhance NO-induced Akt pathway activation. Moreover, TIMP-1 silencing by antisense technology blocked NO-induced PI3k/Akt/BAD phosphorylation in cultured MDA-MB-231 human breast cancer cells. TIMP-1 protein nitration and TIMP-1/CD63 co-immunoprecipitation was observed at NO concentrations that induced PI3k/Akt/BAD pro-survival signaling. In the survival analysis, elevated tumor TIMP-1 predicted poor patient survival. This association appears to be mainly restricted to tumors with high NOS2 protein. In contrast, TIMP-1 did not predict poor survival in patient tumors with low NOS2 expression. In summary, our findings suggest that tumors with high TIMP-1 and NOS2 behave more aggressively by mechanisms that favor Akt pathway activation.

First-generation interaction maps of Src homology 2 (SH2) domains with receptor tyrosine kinase (RTK) phosphosites have previously been generated using protein microarray (PM) technologies. Here, we developed a large-scale fluorescence polarization (FP) methodology that was able to characterize interactions between SH2 domains and ErbB receptor phosphosites with higher fidelity and sensitivity than was previously achieved with PMs. We used the FP assay to query the interaction of synthetic phosphopeptides corresponding to 89 ErbB receptor intracellular tyrosine sites against 93 human SH2 domains and 2 phosphotyrosine binding (PTB) domains. From 358,944 polarization measurements, the affinities for 1,405 unique biological interactions were determined, 83% of which are novel. In contrast to data from previous reports, our analyses suggested that ErbB2 was not more promiscuous than the other ErbB receptors. Our results showed that each receptor displays unique preferences in the affinity and location of recruited SH2 domains that may contribute to differences in downstream signaling potential. ErbB1 was enriched versus the other receptors for recruitment of domains from RAS GEFs whereas ErbB2 was enriched for recruitment of domains from tyrosine and phosphatidyl inositol phosphatases. ErbB3, the kinase inactive ErbB receptor family member, was predictably enriched for recruitment of domains from phosphatidyl inositol kinases and surprisingly, was enriched for recruitment of domains from tyrosine kinases, cytoskeletal regulatory proteins, and RHO GEFs but depleted for recruitment of domains from phosphatidyl inositol phosphatases. Many novel interactions were also observed with phosphopeptides corresponding to ErbB receptor tyrosines not previously reported to be phosphorylated by mass spectrometry, suggesting the existence of many biologically relevant RTK sites that may be phosphorylated but below the detection threshold of standard mass spectrometry procedures. This dataset represents a rich source of testable hypotheses regarding the biological mechanisms of ErbB receptors.

Cub domain containing protein 1 (CDCP1) is strongly expressed in tumors derived from lung, colon, ovary, or kidney. It is a membrane protein that is phosphorylated and then bound by Src family kinases. Although expression and phosphorylation of CDCP1 have been investigated in many tumor cell lines, the CDCP1 features responsible for transformation have not been fully evaluated. This is in part due to the lack of an experimental system in which cellular transformation depends on expression of exogenous CDCP1 and Src. Here we use retrovirus mediated co-overexpression of c-Src and CDCP1 to induce focus formation of NIH3T3 cells. Employing different mutants of CDCP1 we show that for a full transformation capacity, the intact amino- and carboxy-termini of CDCP1 are essential. Mutation of any of the core intracellular tyrosine residues (Y734, Y743, or Y762) abolished transformation, and mutation of a palmitoylation motif (C689,690G) strongly reduced it. Src kinase binding to CDCP1 was not required since Src with a defective SH2 domain generated even more CDCP1 dependent foci whereas Src myristoylation was necessary. Taken together, the focus formation assay allowed us to define structural requirements of CDCP1/Src dependent transformation and to characterize the interaction of CDCP1 and Src.

All eukaryotes have the ability to detect and respond to environmental and hormonal signals. In many cases these signals evoke cellular changes that are incompatible and must therefore be orchestrated by the responding cell. In the yeast Saccharomyces cerevisiae, hyperosmotic stress and mating pheromones initiate signaling cascades that each terminate with a MAP kinase, Hog1 and Fus3, respectively. Despite sharing components, these pathways are initiated by distinct inputs and produce distinct cellular behaviors. To understand how these responses are coordinated, we monitored the pheromone response during hyperosmotic conditions. We show that hyperosmotic stress limits pheromone signaling in at least three ways. First, stress delays the expression of pheromone-induced genes. Second, stress promotes the phosphorylation of a protein kinase, Rck2, and thereby inhibits pheromone-induced protein translation. Third, stress promotes the phosphorylation of a shared pathway component, Ste50, and thereby dampens pheromone-induced MAPK activation. Whereas all three mechanisms are dependent on an increase in osmolarity, only the phosphorylation events require Hog1. These findings reveal how an environmental stress signal is able to postpone responsiveness to a competing differentiation signal, by acting on multiple pathway components, in a coordinated manner., Author Summary All cells can detect and respond to signals in their environment. The ability to interpret these signals with accuracy is needed for proper growth and differentiation. Moreover, cells must prioritize responses when confronted with competing signals. However the molecular mechanisms that govern signal prioritization are poorly understood. To address this question, we studied two signaling pathways in the genetic model organism budding yeast. Specifically we focused on the pheromone mating (differentiation) pathway and the high osmolarity glycerol (stress response) pathway. These pathways respond differently to each stimulus despite sharing pathway components. We find that cells must first adapt to stress before they can mate. At early times, the stress response cross-inhibits and dampens the pheromone response to suspend mating differentiation. Once cells adapt, the stress response ends and the differentiation program resumes. All signaling pathways that regulate cell fate decisions are interconnected to varying degrees. Our study highlights the importance of proper signal coordination in cell fate decisions, and it reveals new mechanisms that govern signal coordination within complex signaling networks.

Mammalian Target of Rapamycin Complex 1 (mTORC1) is activated by growth factor-regulated phosphoinositide 3-kinase (PI3K)/Akt/Rheb signalling and extracellular amino acids (AAs) to promote growth and proliferation. These AAs induce translocation of mTOR to late endosomes and lysosomes (LELs), subsequent activation via mechanisms involving the presence of intralumenal AAs, and interaction between mTORC1 and a multiprotein assembly containing Rag GTPases and the heterotrimeric Ragulator complex. However, the mechanisms by which AAs control these different aspects of mTORC1 activation are not well understood. We have recently shown that intracellular Proton-assisted Amino acid Transporter 1 (PAT1)/SLC36A1 is an essential mediator of AA-dependent mTORC1 activation. Here we demonstrate in Human Embryonic Kidney (HEK-293) cells that PAT1 is primarily located on LELs, physically interacts with the Rag GTPases and is required for normal AA-dependent mTOR relocalisation. We also use the powerful in vivo genetic methodologies available in Drosophila to investigate the regulation of the PAT1/Rag/Ragulator complex. We show that GFP-tagged PATs reside at both the cell surface and LELs in vivo, mirroring PAT1 distribution in several normal mammalian cell types. Elevated PI3K/Akt/Rheb signalling increases intracellular levels of PATs and synergistically enhances PAT-induced growth via a mechanism requiring endocytosis. In light of the recent identification of the vacuolar H+-ATPase as another Rag-interacting component, we propose a model in which PATs function as part of an AA-sensing engine that drives mTORC1 activation from LEL compartments. © 2012 Ögmundsdóttir et al.

Lung cancer is the most common cause of cancer-related death worldwide. A poor overall survival rate of 16% necessitates the need for novel treatment strategies. Mouse models of lung cancer are important tools for analyzing the significance of somatic mutations in the initiation and progression of lung cancer. Of additional importance, however, are animal models of virally induced cancers. JSRV is a simple betaretrovirus that causes contagious lung cancer in sheep known as ovine pulmonary adenocarcinoma and closely resembles human lung adenocarcinoma. Previously we showed that expression of the JSRV envelope (Env) from an AAV vector induced lung tumors in immunodeficient mice, but not in immunocompetent mice. Because of the importance of studying lung cancer in the context of an intact immune system we sought to improve our mouse model. In this report, we employed the use of a strong JSRV enhancer-promoter combination to express Env at high levels and demonstrate for the first time, lung tumor induction in immunocompetent mice. This occurred despite a robust Env-specific antibody-mediated immune response. The PI3K/Akt and MAPK pathways were activated in both immunocompetent and immunodeficient mice, however, differential activation of PTEN, GSKα, p70S6K, p38MAPK, ATF2 and STAT5 was observed. A JSRV Env lung tumor-derived cell line was shown to have a similar signal transduction activation profile as Env-induced lung tumors in C57BL/6 mice. Given the similarities between our model and pulmonary adenocarcinomas in humans, and the ease with which tumors can be induced in any transgenic mouse, this system can be used to uncover novel mechanisms involved lung tumorigenesis.

Insulin-like growth factor-I (IGF-I) is an essential growth factor that regulates the processes necessary for cell proliferation, differentiation, and survival. The Igf1 gene encodes mature IGF-I and a carboxy-terminal extension called the E-peptide. In rodents, alternative splicing and post-translational processing produce two E-peptides (EA and EB). EB has been studied extensively and has been reported to promote cell proliferation and migration independently of IGF-I and its receptor (IGF-IR), but the mechanism by which EB causes these actions has not been identified. Further, the properties of EA have not been evaluated. Therefore, the goals of this study were to determine if EA and EB possessed similar activity and if these actions were IGF-IR independent. We utilized synthetic peptides for EA, EB, and a scrambled control to examine cellular responses. Both E-peptides increased MAPK signaling, which was blocked by pharmacologic IGF-IR inhibition. Although the E-peptides did not directly induce IGF-IR phosphorylation, the presence of either E-peptide increased IGF-IR activation by IGF-I, and this was achieved through enhanced cell surface bioavailability of the receptor. To determine if E-peptide biological actions required the IGF-IR, we took advantage of the murine C2C12 cell line as a platform to examine the key steps of skeletal muscle proliferation, migration and differentiation. EB increased myoblast proliferation and migration while EA delayed differentiation. The proliferation and migration effects were inhibited by MAPK or IGF-IR signaling blockade. Thus, in contrast to previous studies, we find that E-peptide signaling, mitogenic, and motogenic effects are dependent upon IGF-IR. We propose that the E-peptides have little independent activity, but instead affect growth via modulating IGF-I signaling, thereby increasing the complexity of IGF-I biological activity.

Glucagon-like peptide-1 (GLP-1) released from intestinal L cells in response to nutrients has many physiological effects but particularly enhances glucose-dependent insulin release through the GLP-1 receptor (GLP-1R). GLP-1 7-36 amide, the predominant circulating active form of GLP-1, is rapidly truncated by dipeptidyl peptidase-4 to GLP-1 9-36 amide, which is generally considered inactive. Given its physiological roles, the GLP-1R is targeted for treatment of type 2 diabetes. Recently 'compound 2' has been described as both an agonist and positive allosteric modulator of GLP-1 7-36 amide affinity, but not potency, at the GLP-1R. Importantly, we demonstrated previously that exendin 9-39, generally considered a GLP-1R antagonist, enhances compound 2 efficacy (or vice versa) at the GLP-1R. Given that GLP-1 9-36 amide is the major circulating form of GLP-1 post-prandially and is a low affinity weak partial agonist or antagonist at the GLP-1R, we investigated interaction between this metabolite and compound 2 in a cell line with recombinant expression of the human GLP-1R and the rat insulinoma cell line, INS-1E, with native expression of the GLP-1R. We show compound 2 markedly enhances efficacy and potency of GLP-1 9-36 amide for key cellular responses including AMP generation, Ca(2+) signaling and extracellular signal-regulated kinase. Thus, metabolites of peptide hormones including GLP-1 that are often considered inactive may provide a means of manipulating key aspects of receptor function and a novel therapeutic strategy.

Small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran) regulate key cellular processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. A great deal of experimental evidence supports the existence of signaling cascades and feedback loops within and among the small GTPase subfamilies suggesting that these proteins function in a coordinated and cooperative manner. The interplay occurs largely through association with bi-partite regulatory and effector proteins but can also occur through the active form of the small GTPases themselves. In order to understand the connectivity of the small GTPases signaling routes, a systems-level approach that analyzes data describing direct and indirect interactions was used to construct the small GTPases protein interaction network. The data were curated from the Search Tool for the Retrieval of Interacting Genes (STRING) database and include only experimentally validated interactions. The network method enables the conceptualization of the overall structure as well as the underlying organization of the protein-protein interactions. The interaction network described here is comprised of 778 nodes and 1943 edges and has a scale-free topology. Rac1, Cdc42, RhoA, and HRas are identified as the hubs. Ten sub-network motifs are also identified in this study with themes in apoptosis, cell growth/proliferation, vesicle traffic, cell adhesion/junction dynamics, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase response, transcription regulation, receptor-mediated endocytosis, gene silencing, and growth factor signaling. Bottleneck proteins that bridge signaling paths and proteins that overlap in multiple small GTPase networks are described along with the functional annotation of all proteins in the network.

BackgroundExtensive DNA damage leads to apoptosis. Histones play a central role in DNA damage sensing and may mediate signals of genotoxic damage to cytosolic effectors including mitochondria.Methodology/principal findingsWe have investigated the effects of histones on mitochondrial function and membrane integrity. We demonstrate that both linker histone H1 and core histones H2A, H2B, H3, and H4 bind strongly to isolated mitochondria. All histones caused a rapid and massive release of the pro-apoptotic intermembrane space proteins cytochrome c and Smac/Diablo, indicating that they permeabilize the outer mitochondrial membrane. In addition, linker histone H1, but not core histones, permeabilized the inner membrane with a collapse of the membrane potential, release of pyridine nucleotides, and mitochondrial fragmentation.ConclusionsWe conclude that histones destabilize the mitochondrial membranes, a mechanism that may convey genotoxic signals to mitochondria and promote apoptosis following DNA damage.

PTP1B is an endoplasmic reticulum (ER) anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC) in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and co-localization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM) associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC). Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research.

BACKGROUND Pressure overload and prolonged angiotensin II (Ang II) infusion elicit cardiac hypertrophy in Ang II receptor 1 (AT(1)) null mouse, whereas Ang II receptor 2 (AT(2)) gene deletion abolishes the hypertrophic response. The roles and signals of the cardiac AT(2) receptor still remain unsettled. Promyelocytic leukemia zinc finger protein (PLZF) was shown to bind to the AT(2) receptor and transmit the hypertrophic signal. Using PLZF knockout mice we directed our studies on the function of PLZF concerning the cardiac specific transcription factor GATA4, and GATA4 targets. METHODOLOGY AND PRINCIPAL FINDINGS PLZF knockout and age-matched wild-type (WT) mice were treated with Ang II, infused at a rate of 4.2 ng·kg(-1)·min(-1) for 3 weeks. Ang II elevated systolic blood pressure to comparable levels in PLZF knockout and WT mice (140 mmHg). WT mice developed prominent cardiac hypertrophy and fibrosis after Ang II infusion. In contrast, there was no obvious cardiac hypertrophy or fibrosis in PLZF knockout mice. An AT(2) receptor blocker given to Ang II-infused wild type mice prevented hypertrophy, verifying the role of AT(2) receptor for cardiac hypertrophy. Chromatin immunoprecipitation and electrophoretic mobility shift assay showed that PLZF bound to the GATA4 gene regulatory region. A Luciferase assay verified that PLZF up-regulated GATA4 gene expression and the absence of PLZF expression in vivo produced a corresponding repression of GATA4 protein. CONCLUSIONS PLZF is an important AT(2) receptor binding protein in mediating Ang II induced cardiac hypertrophy through an AT(2) receptor-dependent signal pathway. The angiotensin II-AT(2)-PLZF-GATA4 signal may further augment Ang II induced pathological effects on cardiomyocytes.

Consequences of expression of the protein tyrosine phosphatase nonreceptor 22 (PTPN22) gain-of-function variant were evaluated in leukocytes from patients with anti-neutrophil cytoplasmic autoantibody (ANCA) disease. The frequency of the gain-of-function allele within the Caucasian patient cohort was 22% (OR 1.45), compared to general American Caucasian population (16.5%, p = 0.03). Examination of the basal phosphatase activity of PTPN22 gain-of-function protein indicated persistently elevated activity in un-stimulated peripheral leukocytes, while basal activity was undetectable in leukocytes from patients without the gain-of-function variant. To examine consequences of persistently high PTPN22 activity, the activation status of ERK and p38 MAPK were analyzed. While moderate levels of activated ERK were observed in controls, it was undetectable in leukocytes expressing PTPN22 gain-of-function protein and instead p38MAPK was up-regulated. IL-10 transcription, reliant on the ERK pathway, was negatively affected. Over the course of disease, patients expressing variant PTPN22 did not show a spike in IL-10 transcription as they entered remission in contrast to controls, implying that environmentally triggered signals were blunted. Sustained activity of PTPN22, due to the gain-of-function mutation, acts as a dominant negative regulator of ERK activity leading to blunted cellular responsiveness to environmental stimuli and expression of protective cytokines.

Entry of Neisseria meningitidis (the meningococcus) into human brain microvascular endothelial cells (HBMEC) is mediated by fibronectin or vitronectin bound to the surface protein Opc forming a bridge to the respective integrins. This interaction leads to cytoskeletal rearrangement and uptake of meningococci. In this study, we determined that the focal adhesion kinase (FAK), which directly associates with integrins, is involved in integrin-mediated internalization of N. meningitidis in HBMEC. Inhibition of FAK activity by the specific FAK inhibitor PF 573882 reduced Opc-mediated invasion of HBMEC more than 90%. Moreover, overexpression of FAK mutants that were either impaired in the kinase activity or were not capable of autophosphorylation or overexpression of the dominant-negative version of FAK (FRNK) blocked integrin-mediated internalization of N. meningitidis. Importantly, FAK-deficient fibroblasts were significantly less invaded by N. meningitidis. Furthermore, N. meningitidis induced tyrosine phosphorylation of several host proteins including the FAK/Src complex substrate cortactin. Inhibition of cortactin expression by siRNA silencing and mutation of critical amino acid residues within cortactin, that encompass Arp2/3 association and dynamin binding, significantly reduced meningococcal invasion into eukaryotic cells suggesting that both domains are critical for efficient uptake of N. meningitidis into eukaryotic cells. Together, these results indicate that N. meningitidis exploits the integrin signal pathway for its entry and that FAK mediates the transfer of signals from activated integrins to the cytoskeleton. A cooperative interplay between FAK, Src and cortactin then enables endocytosis of N. meningitidis into host cells.

Phosphorylation of actin-binding proteins plays a pivotal role in the remodeling of the actin cytoskeleton to regulate cell migration. Palladin is an actin-binding protein that is phosphorylated by growth factor stimulation; however, the identity of the involved protein kinases remains elusive. In this study, we report that palladin is a novel substrate of extracellular signal-regulated kinase (ERK). Suppression of ERK activation by a chemical inhibitor reduced palladin phosphorylation, and expression of active MEK alone was sufficient for phosphorylation. In addition, an in vitro kinase assay demonstrated direct palladin phosphorylation by ERK. We found that Ser77 and Ser197 are essential residues for phosphorylation. Although the phosphorylation of these residues was not required for actin cytoskeletal organization, we found that expression of non-phosphorylated palladin enhanced cell migration. Finally, we show that phosphorylation inhibits the palladin association with Abl tyrosine kinase. Taken together, our results indicate that palladin phosphorylation by ERK has an anti-migratory function, possibly by modulating interactions with molecules that regulate cell migration.

MAPK signal transduction pathways are important regulators of stress responses, cellular growth, and differentiation. In Neurospora, the circadian clock controls rhythms in phosphorylation of the p38-like MAPK (OS-2); however, the mechanism for this regulation is not known. We show that the WCC, a transcription factor and clock component, binds to the os-4 MAPKKK promoter in response to light and rhythmically in constant darkness, peaking in the subjective morning. Deletion of the WCC binding sites in the os-4 promoter disrupts both os-4 mRNA and OS-2 phosphorylation rhythms. The clock also indirectly regulates rhythmic expression of the histidyl-phosphotransferase gene, hpt-1, which peaks in the evening. Anti-phase expression of positive (OS-4) and negative (HPT-1) MAPK pathway regulators likely coordinate to enhance rhythmic MAPK activation to prepare cells to respond to osmotic stress during the day in the natural environment. Consistent with this idea, we show that wild type cells have a clock-dependent morning kinetic advantage in glycerol accumulation after salt stress as compared to evening treatment. Thus, circadian transcriptional control of MAPK pathway components leads to striking time-of-day-specific effects on the signaling status and physiological response of the pathway.

Purpose: Imatinib Mesylate (Gleevec) is a drug that potently counteracts diabetes both in humans and in animal models for human diabetes. We have previously reported that this compound in human pancreatic islets stimulates NF-kappa B signaling and islet cell survival. The aim of this study was to investigate control of NF-kappa B post-translational modifications exerted by Imatinib and whether any such effects are associated with altered islet gene expression and chemokine production in vitro. Procedures: Human islets were either left untreated or treated with Imatinib for different timepoints. I kappa B-alpha and NF-kappa B p65 phosphorylation and methylation were assessed by immunoblot analysis. Islet gene expression was assessed using a commercial Pathway Finder microarray kit and RT-PCR. Islet chemokine production was determined by flow cytometric bead array analysis. Findings: Human islet I kappa B-alpha and Ser276-p65 phosphorylation were increased by a 20 minute Imatinib exposure. Methylation of p65 at position Lys221 was increased after 60 min of Imatinib exposure and persisted for 3 hours. Microarray analysis of islets exposed to Imatinib for 4 hours revealed increased expression of the inflammatory genes IL-4R, TCF5, DR5, I-TRAF, I-CAM, HSP27 and IL-8. The islet release of IL-8 was augmented in islets cultured over night in the presence of Imatinib. Following 30 hours of Imatinib exposure, the cytokine-induced I kappa B-alpha and STAT1 phosphorylation was abolished and diminished, respectively. The cytokine-induced release of the chemokines MIG and IP10 was lower in islets exposed to Imatinib for 30 hours. Conclusion: Imatinib by itself promotes a modest activation of NF-kappa B. However, a prolonged exposure of human islets to Imatinib is associated with a dampened response to cytokines. It is possible that Imatinib induces NF-kappa B preconditioning of islet cells leading to lowered cytokine sensitivity and a mitigated islet inflammation.

The receptor for advanced glycation end products (RAGE) is thought to be involved in the pathogenesis of a broad range of inflammatory, degenerative and hyperproliferative diseases. It binds to diverse ligands and activates multiple intracellular signaling pathways. Despite these pivotal functions, molecular events just downstream of ligand-activated RAGE have been surprisingly unknown. Here we show that the cytoplasmic domain of RAGE is phosphorylated at Ser391 by PKCζ upon binding of ligands. TIRAP and MyD88, which are known to be adaptor proteins for Toll-like receptor-2 and -4 (TLR2/4), bound to the phosphorylated RAGE and transduced a signal to downstream molecules. Blocking of the function of TIRAP and MyD88 largely abrogated intracellular signaling from ligand-activated RAGE. Our findings indicate that functional interaction between RAGE and TLRs coordinately regulates inflammation, immune response and other cellular functions.

Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) serve as Wnt co-receptors for the canonical β-catenin pathway. While LRP6 is essential for embryogenesis, both LRP5 and LRP6 play critical roles for skeletal remodeling, osteoporosis pathogenesis and cancer formation, making LRP5 and LRP6 key therapeutic targets for cancer and disease treatment. LRP5 and LRP6 each contain in the cytoplasmic domain five conserved PPPSPxS motifs that are pivotal for signaling and serve collectively as phosphorylation-dependent docking sites for the scaffolding protein Axin. However existing data suggest that LRP6 is more effective than LRP5 in transducing the Wnt signal. To understand the molecular basis that accounts for the different signaling activity of LRP5 and LRP6, we generated a series of chimeric receptors via swapping LRP5 and LRP6 cytoplasmic domains, LRP5C and LRP6C, and studied their Wnt signaling activity using biochemical and functional assays. We demonstrate that LRP6C exhibits strong signaling activity while LRP5C is much less active in cells. Recombinant LRP5C and LRP6C upon in vitro phosphorylation exhibit similar Axin-binding capability, suggesting that LRP5 and LRP6 differ in vivo at a step prior to Axin-binding, likely at receiving phosphorylation. We identified between the two most carboxyl PPPSPxS motifs an intervening "gap4" region that appears to account for much of the difference between LRP5C and LRP6C, and showed that alterations in this region are sufficient to enhance LRP5 PPPSPxS phosphorylation and signaling to levels comparable to LRP6 in cells. In addition we provide evidence that binding of phosphorylated LRP5 or LRP6 to Axin is likely direct and does not require the GSK3 kinase as a bridging intermediate as has been proposed. Our studies therefore uncover a new and important molecular tuning mechanism for differential regulation of LRP5 and LRP6 phosphorylation and signaling activity.

Our previous studies have shown that benzyl isothiocyanate (BITC) suppresses pancreatic tumor growth by inhibiting STAT-3; however, the exact mechanism of tumor growth suppression was not clear. Here we evaluated the effects and mechanism of BITC on pancreatic tumor angiogenesis. Our results reveal that BITC significantly inhibits neovasularization on rat aorta and Chicken-Chorioallantoic membrane. Furthermore, BITC blocks the migration and invasion of BxPC-3 and PanC-1 pancreatic cancer cells in a dose dependant manner. Moreover, secretion of VEGF and MMP-2 in normoxic and hypoxic BxPC-3 and PanC-1 cells was significantly suppressed by BITC. Both VEGF and MMP-2 play a critical role in angiogenesis and metastasis. Our results reveal that BITC significantly suppresses the phosphorylation of VEGFR-2 (Tyr-1175), and expression of HIF-α. Rho-GTPases, which are regulated by VEGF play a crucial role in pancreatic cancer progression. BITC treatment reduced the expression of RhoC whereas up-regulated the expression of tumor suppressor RhoB. STAT-3 over-expression or IL-6 treatment significantly induced HIF-1α and VEGF expression; however, BITC substantially suppressed STAT-3 as well as STAT-3-induced HIF-1α and VEGF expression. Finally, in vivo tumor growth and matrigel-plug assay show reduced tumor growth and substantial reduction of hemoglobin content in the matrigel plugs and tumors of mice treated orally with 12 µmol BITC, indicating reduced tumor angiogenesis. Immunoblotting of BITC treated tumors show reduced expression of STAT-3 phosphorylation (Tyr-705), HIF-α, VEGFR-2, VEGF, MMP-2, CD31 and RhoC. Taken together, our results suggest that BITC suppresses pancreatic tumor growth by inhibiting tumor angiogenesis through STAT-3-dependant pathway.

Background The complement component C3a induces degranulation in human mast cells via the activation of cell surface G protein coupled receptors (GPCR; C3aR). For most GPCRs, agonist-induced receptor phosphorylation leads to the recruitment of β-arrestin-1/β-arrestin-2; resulting in receptor desensitization and internalization. Activation of GPCRs also leads to ERK1/2 phosphorylation via two temporally distinct pathways; an early response that reflects G protein activation and a delayed response that is G protein independent but requires β-arrestins. The role of β-arrestins on C3aR activation/regulation in human mast cells, however, remains unknown. Methodology/Principal Findings We utilized lentivirus short hairpin (sh)RNA to stably knockdown the expression of β-arrestin-1 and β-arrrestin-2 in human mast cell lines, HMC-1 and LAD2 that endogenously expresses C3aR. Silencing β-arrestin-2 attenuated C3aR desensitization, blocked agonist-induced receptor internalization and rendered the cells responsive to C3a for enhanced NF-κB activity as well as chemokine generation. By contrast, silencing β-arrestin-1 had no effect on these responses but resulted in a significant decrease in C3a-induced mast cell degranulation. In shRNA control cells, C3a caused a transient ERK1/2 phosphorylation, which peaked at 5 min but disappeared by 10 min. Knockdown of β-arrestin-1, β-arrestin-2 or both enhanced the early response to C3a and rendered the cells responsive for ERK1/2 phosphorylation at later time points (10–30 min). Treatment of cells with pertussis toxin almost completely blocked both early and delayed C3a-induced ERK1/2 phosphorylation in β-arrestin1/2 knockdown cells. Conclusion/Significance This study demonstrates distinct roles for β-arrestins-1 and β-arrestins-2 on C3aR desensitization, internalization, degranulation, NF-κB activation and chemokine generation in human mast cells. It also shows that both β-arrestin-1 and β-arrestin-2 play a novel and shared role in inhibiting G protein-dependent ERK1/2 phosphorylation. These findings reveal a new level of complexity for C3aR regulation by β-arrestins in human mast cells.

Streptococcus pyogenes (Group A Streptococcus, GAS) is an important human commensal that occasionally causes localized infections and less frequently causes severe invasive disease with high mortality rates. How GAS regulates expression of factors used to colonize the host and avoid immune responses remains poorly understood. Intercellular communication is an important means by which bacteria coordinate gene expression to defend against host assaults and competing bacteria, yet no conserved cell-to-cell signaling system has been elucidated in GAS. Encoded within the GAS genome are four rgg-like genes, two of which (rgg2 and rgg3) have no previously described function. We tested the hypothesis that rgg2 or rgg3 rely on extracellular peptides to control target-gene regulation. We found that Rgg2 and Rgg3 together tightly regulate two linked genes encoding new peptide pheromones. Rgg2 activates transcription of and is required for full induction of the pheromone genes, while Rgg3 plays an antagonistic role and represses pheromone expression. The active pheromone signals, termed SHP2 and SHP3, are short and hydrophobic (DI[I/L]IIVGG), and, though highly similar in sequence, their ability to disrupt Rgg3-DNA complexes were observed to be different, indicating that specificity and differential activation of promoters are characteristics of the Rgg2/3 regulatory circuit. SHP-pheromone signaling requires an intact oligopeptide permease (opp) and a metalloprotease (eep), supporting the model that pro-peptides are secreted, processed to the mature form, and subsequently imported to the cytoplasm to interact directly with the Rgg receptors. At least one consequence of pheromone stimulation of the Rgg2/3 pathway is increased biogenesis of biofilms, which counteracts negative regulation of biofilms by RopB (Rgg1). These data provide the first demonstration that Rgg-dependent quorum sensing functions in GAS and substantiate the role that Rggs play as peptide receptors across the Firmicute phylum., Author Summary Group A Streptococcus (GAS, Streptococcus pyogenes) is a pathogen responsible for a wide variety of diseases including pharyngitis and toxic shock, but is also commonly carried in the human population asymptomatically. What causes the switch between carrier and pathogenic states remains poorly understood. Cell-to-cell signaling in bacteria, also known as quorum sensing, is an important mechanism for virulence gene regulation in many pathogens, yet no conserved quorum sensing pathway has been elucidated in GAS. Here we identify and characterize a new GAS quorum-sensing circuit that consists of two regulators that respond to two secreted peptide signals. The regulators are members of a class of generally poorly studied proteins (known as Rgg) found throughout Gram-positive bacteria. The mature peptide signals, here termed pheromones for their ability to elicit responses in members of the population, enhance the production of biofilms, which have been associated with nasopharyngeal colonization, antibiotic-treatment failures, and necrotic infections. We found that exogenous addition of synthetically-produced pheromone also induced biofilm growth, demonstrating that GAS behavior can be manipulated by artificial signals and offering the possibility for alternative therapeutic treatments.

Background Acidification of the cytoplasm and the extracellular environment is associated with many physiological and pathological conditions, such as intense exercise, hypoxia and tumourigenesis. Acidification affects important cellular functions including protein synthesis, growth, and proliferation. Many of these vital functions are controlled by mTORC1, a master regulator protein kinase that is activated by various growth-stimulating signals and inactivated by starvation conditions. Whether mTORC1 can also respond to changes in extracellular or cytoplasmic pH and play a role in limiting anabolic processes in acidic conditions is not known. Methodology/Findings We examined the effects of acidifying the extracellular medium from pH 7.4 to 6.4 on human breast carcinoma MCF-7 cells and immortalized mouse embryo fibroblasts. Decreasing the extracellular pH caused intracellular acidification and rapid, graded and reversible inhibition of mTORC1, assessed by measuring the phosphorylation of the mTORC1 substrate S6K. Fibroblasts deleted of the tuberous sclerosis complex TSC2 gene, a major negative regulator of mTORC1, were unable to inhibit mTORC1 in acidic extracellular conditions, showing that the TSC1–TSC2 complex is required for this response. Examination of the major upstream pathways converging on the TSC1–TSC2 complex showed that Akt signaling was unaffected by pH but that the Raf/MEK/ERK pathway was inhibited. Inhibition of MEK with drugs caused only modest mTORC1 inhibition, implying that other unidentified pathways also play major roles. Conclusions This study reveals a novel role for the TSC1/TSC2 complex and mTORC1 in sensing variations in ambient pH. As a common feature of low tissue perfusion, low glucose availability and high energy expenditure, acidic pH may serve as a signal for mTORC1 to downregulate energy-consuming anabolic processes such as protein synthesis as an adaptive response to metabolically stressful conditions.

BACKGROUND: A central aspect of development and disease is the control of cell proliferation through regulation of the mitotic cycle. Cell cycle progression and directionality requires an appropriate balance of positive and negative regulators whose expression must fluctuate in a coordinated manner. p19INK4d, a member of the INK4 family of CDK inhibitors, has a unique feature that distinguishes it from the remaining INK4 and makes it a likely candidate for contributing to the directionality of the cell cycle. p19INK4d mRNA and protein levels accumulate periodically during the cell cycle under normal conditions, a feature reminiscent of cyclins. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we demonstrate that p19INK4d is transcriptionally regulated by E2F1 through two response elements present in the p19INK4d promoter. Ablation of this regulation reduced p19 levels and restricted its expression during the cell cycle, reflecting the contribution of a transcriptional effect of E2F1 on p19 periodicity. The induction of p19INK4d is delayed during the cell cycle compared to that of cyclin E, temporally separating the induction of these proliferative and antiproliferative target genes. Specific inhibition of the E2F1-p19INK4d pathway using triplex-forming oligonucleotides that block E2F1 binding on p19 promoter, stimulated cell proliferation and increased the fraction of cells in S phase. CONCLUSIONS/SIGNIFICANCE: The results described here support a model of normal cell cycle progression in which, following phosphorylation of pRb, free E2F induces cyclin E, among other target genes. Once cyclinE/CDK2 takes over as the cell cycle driving kinase activity, the induction of p19 mediated by E2F1 leads to inhibition of the CDK4,6-containing complexes, bringing the G1 phase to an end. This regulatory mechanism constitutes a new negative feedback loop that terminates the G1 phase proliferative signal, contributing to the proper coordination of the cell cycle and provides an additional mechanism to limit E2F activity.