Gain-of-function hot spot mutant p53R248Q regulation of integrin/FAK/ERK signaling in esophageal squamous cell carcinoma

PurposeTP53, encoding the protein p53, is among the most frequently mutated genes in all cancers. A high frequency of 60 – 90% mutations is seen in esophageal squamous cell carcinoma (ESCC) patients. Certain p53 mutants show gain-of-function (GoF) oncogenic features unrelated to its wild type functions.MethodsThis study functionally characterized a panel of p53 mutants in individual ESCC cell lines and assayed for GoF oncogenic properties.ResultsThe ESCC cell line with endogenous p53^R248Q expression showed suppressed tumor growth in an immunocompromised mouse model and suppressed colony growth in in vitro three-dimensional culture, when depleted of the endogenous p53 protein expression. This suppression is accompanied by suppressed cell cycle progression, along with reduced integrin expression and decreased focal adhesion kinase and extracellular-regulated protein kinase signaling and can be compensated by expression of a constitutively active mitogen-activated protein. P53^R248Q enhances cell proliferation upon glutamine deprivation, as compared to other non-GoF mutants.ConclusionsIn summary, study of the functional contributions of endogenous p53 mutants identified a novel GoF mechanism through which a specific p53 mutant exerts oncogenic features and contributes to ESCC tumorigenesis.     

Chromatin,And Gene Regulation In Eukaryotic Cells At The Transcriptional Leve

Phytochromes are environmental sensors, historically thought of as red/far-red photoreceptors in plants. Their photoperception occurs through a covalently linked tetrapyrrole chromophore, which undergoes a light-dependent conformational change propagated through the protein to a variable output domain. The phytochrome composition is modular, typically consisting of a PAS-GAF-PHY architecture for the N-terminal photosensory core. A collection of three-dimensional structures has uncovered key features, including an unusual figure-of-eight knot, an extension reaching from the PHY domain to the chromophore-binding GAF domain, and a centrally located, long α-helix hypothesized to be crucial for intramolecular signaling. Continuing identification of phytochromes in microbial systems has expanded the assigned sensory abilities of this family out of the red and into the yellow, green, blue, and violet portions of the spectrum. Furthermore, phytochromes acting not as photoreceptors but as redox sensors have been recognized. In addition, architectures other than PAS-GAF-PHY are known, thus revealing phytochromes to be a varied group of sensory receptors evolved to utilize their modular design to perceive a signal and respond accordingly. This review focuses on the structures of bacterial phytochromes and implications for signal transmission. We also discuss the small but growing set of bacterial phytochromes for which a physiological function has been ascertained.     

Reactive oxygen species in cancer

Abstract

Elevated rates of reactive oxygen species (ROS) have been detected in almost all cancers, where they promote many aspects of tumour development and progression. However, tumour cells also express increased levels of antioxidant proteins to detoxify from ROS, suggesting that a delicate balance of intracellular ROS levels is required for cancer cell function. Further, the radical generated, the location of its generation, as well as the local concentration is important for the cellular functions of ROS in cancer. A challenge for novel therapeutic strategies will be the fine tuning of intracellular ROS signalling to effectively deprive cells from ROS-induced tumour promoting events, towards tipping the balance to ROS-induced apoptotic signalling. Alternatively, therapeutic antioxidants may prevent early events in tumour development, where ROS are important. However, to effectively target cancer cells specific ROS-sensing signalling pathways that mediate the diverse stress-regulated cellular functions need to be identified. This review discusses the generation of ROS within tumour cells, their detoxification, their cellular effects, as well as the major signalling cascades they utilize, but also provides an outlook on their modulation in therapeutics.     

Methylation-mediated regulation of E2F1 in DNA damage-induced cell death

E2F1 promotes DNA damage-induced apoptosis and the post-translational modifications of E2F1 play an important role in the regulation of E2F1-mediated cell death. Here, we found that Set9 and LSD1 regulate E2F1-mediated apoptosis upon DNA damage. Set9 methylates E2F1 at lysine 185, a conserved residue in the DNA-binding domain of E2F family proteins. The methylation of E2F1 by Set9 leads to the stabilization of E2F1 and up-regulation of its proapoptotic target genes p73 and Bim, and thereby induces E2F1-mediated apoptosis in response to genotoxic agents. We also found that LSD1 demethylates E2F1 at lysine 185 and reduces E2F1-mediated cell death. The identification of the methylation/demethylation of E2F1 by Set9/LSD1 suggests that E2F1 is dynamically regulated by epigenetic enzymes in response to DNA damage.     

The Expression of Urotensin II Receptor (U2R) is Up‐regulated by Interferon‐γ

Abstract

Urotensin‐II (U‐II) was identified as the natural ligand of the G protein‐coupled receptor GPR14, which has been correspondingly renamed Urotensin‐II receptor (U2R). The tissue distribution of U2R and the pharmacological effects of U‐II suggest a novel neurohormonal system with potent cardiovascular effects. We here report the human rhabdomyosarcoma cell line TE‐671 as the first natural and endogenous source of functional U2R in an immortalized cell line. In TE‐671 cells, U‐II stimulated extracellular signal regulated kinase phosphorylation and increased c‐fos mRNA expression. Furthermore, we demonstrate that the expression of U2R mRNA and functional U‐II high affinity binding sites are serum‐responsive and that they are specifically up‐regulated by interferon γ (IFNγ). We propose that IFNγ contributes to the previously observed increase of U2R density in the heart tissue of congestive heart failure (CHF) patients and we suggest that U2R up‐regulation, as a consequence of an inflammatory response, could lead to a clinical worsening of this disease.     

Lysophosphatidic acid signaling in ovarian cancer

Abstract

Lysophosphatidic acid (LPA) is a bioactive phospholipid that is involved in signal transduction between cells. Plasma and ascites levels of LPA are increased in ovarian cancer patients even in the early stages and thus LPA is considered as a potential diagnostic marker for this disease. This review presents the current knowledge regarding LPA signaling in epithelial ovarian cancer. LPA stimulates proliferation, migration and invasion of ovarian cancer cells through regulation of vascular endothelial growth factor, matrix metalloproteinases, urokinase plasminogen activator, interleukin-6, interleukin-8, CXC motif chemokine ligand 12/CXC receptor 4, COX2, cyclin D1, Hippo-Yap and growth-regulated oncogene α concentrations. In this article, all of these targets and signal pathways involved in LPA influence are described.     

Lysophosphatidic acid-stimulated phosphorylation of PKD2 is mediated by PI3K p110β and PKCδ in myoblasts

Abstract

Context: G-protein coupled receptor (GPCR) signaling in skeletal muscle is incompletely understood; in particular, the signaling pathways that regulate GPCR-mediated signaling in skeletal muscle are only beginning to be established. Lysophosphatidic acid (LPA) is a GPCR agonist that has previously been shown to activate protein kinase D (PKD) in non-muscle cells; however, whether PKD is activated in response to LPA in skeletal muscle myoblasts, and the identities of signaling intermediates that regulate this activation, have not been defined. Objective: To determine whether PKD is activated in response to LPA administration in myoblasts, and to define the signaling pathways that mediate LPA-stimulated PKD phosphorylation. Methods: C2C12 myoblasts were treated with LPA and signaling pathways examined by means of Western immunoblotting and real-time PCR (RT-PCR). Pharmacological inhibition and RNA-interference were used to target specific molecules to determine their involvement in LPA-induced PKD phosphorylation. Results: Treatment of myoblasts with exogenous LPA revealed that PI3K p110β mediated PKD phosphorylation at Ser 748 and at Ser 916 through kinase-dependent and kinase-independent mechanisms. Loss of PKCδ, but not the loss of PKCα, prevented LPA-induced PKD phosphorylation. The PKD isoform responsive to LPA treatment was identified as PKD2. Conclusion: These results indicate that LPA-stimulated PKD2 phosphorylation requires PKCδ and non-catalytic actions of PI3K p110β, and provide new information with respect to GPCR-mediated signal transduction in myoblasts.     

The hydrophobic amino acid cluster at the cytoplasmic end of transmembrane helix III modulates the coupling of the ß1-adrenergic receptor to Gs

Abstract

A cluster of hydrophobic amino acids at the cytoplasmic end of trans-membranal helix III (TM-III) is a common feature among class-A of G protein-coupled receptors (GPCR). We mutagenized alanine 159^3.53 to glutamic acid and isoleucine160^3.54 to arginine (A159E/I160R) in TM-III of the human ß₁-adrenergic receptor (ß₁-AR) to disrupt the function of the hydrophobic cluster. Structurally, the combined mutations of A159E/I160R caused an almost 90° tilt in the rotation of Arg156^3.50 in the E/DRY motif of TM-III and displaced Tyr166^3.60 in intracellular loop 2. The A159E/I160R ß₁-AR was uncoupled from G_s as determined by cyclic AMP/adenylyl cyclase assays and by FRET-based proximity measurements between the ß₁-AR and G_sα. Isoproterenol induced ß-arrestin trafficking in cells expressing both the wild-type ß₁-AR and the A159E/I160R ß₁-AR. Isoproterenol markedly increased the phosphorylation of ERK_1/2 in cells expressing the WT ß₁-AR and this effect was dependent on the activation of the G_s-cyclic AMP-dependent protein kinase?→?Rap?→?B-raf axis. However, in cells bearing the A159E/I160R ß₁-AR, isoproterenol failed to increase the phosphorylation of ERK_1/2. These results indicate that mutations in the G_sα-binding pocket of the GPCR interfered with receptor coupling to G_s and with its downstream signaling cascades.     

Looking beyond the Wnt pathway for the deep nature of β‐catenin

After two decades of stardom, one would think that β‐catenin has revealed all of its most intimate details. Yet the essence of its duality has remained mysterious—how can a single protein both be the core link between cadherins and the cytoskeleton, and the nuclear messenger for Wnt signalling? On the basis of the available evidence and on molecular and evolutionary considerations, I propose that β‐catenin was a born nuclear transport receptor, which by interacting with adhesion molecules acquired the property to coordinate nuclear functions with cell–cell adhesion. While Wnt signalling diverted this activity, the original pathway might still function in modern eukaryotes.     

Force Measurement Tools to Explore Cadherin Mechanotransduction

Abstract

Cell–cell adhesions serve to mechanically couple cells, allowing for long-range transmission of forces across cells in development, disease, and homeostasis. Recent work has shown that such contacts also play a role in transducing mechanical cues into a wide variety of cellular behaviors important to tissue function. As such, understanding the mechanical regulation of cells through their adhesion molecules has become a point of intense focus. This review will highlight the existing and emerging technologies and models that allow for exploration of cadherin-based adhesions as sites of mechanotransduction.