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  • Distinct control of PERIOD2 degradation and circadian rhythms by the oncoprotein and ubiquitin ligase MDM2
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-13
    JingJing Liu, Xianlin Zou, Tetsuya Gotoh, Anne M. Brown, Liang Jiang, Esther L. Wisdom, Jae Kyoung Kim, Carla V. Finkielstein

    The circadian clock relies on posttranslational modifications to set the timing for degradation of core regulatory components, which drives clock progression. Ubiquitin-modifying enzymes that target clock components for degradation mainly recognize phosphorylated substrates. Degradation of the circadian clock component PERIOD 2 (PER2) is mediated by its phospho-specific recognition by β-transducin repeat–containing proteins (β-TrCPs), which are F-box–containing proteins that function as substrate recognition subunits of the SCFβ-TRCP ubiquitin ligase complex. However, this mode of regulating PER2 stability falls short of explaining the persistent oscillatory phenotypes reported in biological systems lacking functional elements of the phospho-dependent PER2 degradation machinery. We identified PER2 as a previously uncharacterized substrate for the ubiquitin ligase mouse double minute 2 homolog (MDM2) and found that MDM2 targeted PER2 for degradation in a manner independent of PER2 phosphorylation. Deregulation of MDM2 plays a major role in oncogenesis by contributing to the accumulation of genomic and epigenomic alterations that favor tumor development. MDM2-mediated PER2 turnover was important for defining the circadian period length in mammalian cells, a finding that emphasizes the connection between the circadian clock and cancer. Our results not only broaden the range of specific substrates of MDM2 beyond the cell cycle to include circadian components but also identify a previously unknown regulator of the clock as a druggable node that is often found to be deregulated during tumorigenesis.

    更新日期:2018-11-14
  • The basics of mechanotransduction
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-13
    Erin R. Williams

    Basic residues in the transmembrane domain of the T cell receptor α chain promote its association with CD3 signaling chains.

    更新日期:2018-11-14
  • A cytoskeletal anchor connects ischemic mitochondrial fission to myocardial senescence
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-13
    Michael J. Boyer, Satoru Eguchi

    The interplay between the actin cytoskeleton and mitochondria has been implicated in cell and tissue homeostasis and physiological function. In this issue of Science Signaling, Nishimura et al. demonstrate that inhibiting the interaction of filamin A, an actin cytoskeleton regulator, with Drp1, a modulator of mitochondrial dynamics, attenuates mitochondrial hyperfission and cardiomyocyte senescence after myocardial infarction.

    更新日期:2018-11-14
  • The transmembrane adaptor protein NTAL limits mast cell chemotaxis toward prostaglandin E2
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-13
    Ivana Halova, Monika Bambouskova, Lubica Draberova, Viktor Bugajev, Petr Draber

    Chemotaxis of mast cells is one of the crucial steps in their development and function. Non–T cell activation linker (NTAL) is a transmembrane adaptor protein that inhibits the activation of mast cells and B cells in a phosphorylation-dependent manner. Here, we studied the role of NTAL in the migration of mouse mast cells stimulated by prostaglandin E2 (PGE2). Although PGE2 does not induce the tyrosine phosphorylation of NTAL, unlike IgE immune complex antigens, we found that loss of NTAL increased the chemotaxis of mast cells toward PGE2. Stimulation of mast cells that lacked NTAL with PGE2 enhanced the phosphorylation of AKT and the production of phosphatidylinositol 3,4,5-trisphosphate. In resting NTAL-deficient mast cells, phosphorylation of an inhibitory threonine in ERM family proteins accompanied increased activation of β1-containing integrins, which are features often associated with increased invasiveness in tumors. Rescue experiments indicated that only full-length, wild-type NTAL restored the chemotaxis of NTAL-deficient cells toward PGE2. Together, these data suggest that NTAL is a key inhibitor of mast cell chemotaxis toward PGE2, which may act through the RHOA/ERM/β1-integrin and PI3K/AKT axes.

    更新日期:2018-11-14
  • Hypoxia-induced interaction of filamin with Drp1 causes mitochondrial hyperfission–associated myocardial senescence
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-13
    Akiyuki Nishimura, Tsukasa Shimauchi, Tomohiro Tanaka, Kakeru Shimoda, Takashi Toyama, Naoyuki Kitajima, Tatsuya Ishikawa, Naoya Shindo, Takuro Numaga-Tomita, Satoshi Yasuda, Yoji Sato, Koichiro Kuwahara, Yoshito Kumagai, Takaaki Akaike, Tomomi Ide, Akio Ojida, Yasuo Mori, Motohiro Nishida

    Defective mitochondrial dynamics through aberrant interactions between mitochondria and actin cytoskeleton is increasingly recognized as a key determinant of cardiac fragility after myocardial infarction (MI). Dynamin-related protein 1 (Drp1), a mitochondrial fission–accelerating factor, is activated locally at the fission site through interactions with actin. Here, we report that the actin-binding protein filamin A acted as a guanine nucleotide exchange factor for Drp1 and mediated mitochondrial fission–associated myocardial senescence in mice after MI. In peri-infarct regions characterized by mitochondrial hyperfission and associated with myocardial senescence, filamin A colocalized with Drp1 around mitochondria. Hypoxic stress induced the interaction of filamin A with the GTPase domain of Drp1 and increased Drp1 activity in an actin-binding–dependent manner in rat cardiomyocytes. Expression of the A1545T filamin mutant, which potentiates actin aggregation, promoted mitochondrial hyperfission under normoxia. Furthermore, pharmacological perturbation of the Drp1–filamin A interaction by cilnidipine suppressed mitochondrial hyperfission–associated myocardial senescence and heart failure after MI. Together, these data demonstrate that Drp1 association with filamin and the actin cytoskeleton contributes to cardiac fragility after MI and suggests a potential repurposing of cilnidipine, as well as provides a starting point for innovative Drp1 inhibitor development.

    更新日期:2018-11-14
  • Biased agonists of the chemokine receptor CXCR3 differentially control chemotaxis and inflammation
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-06
    Jeffrey S. Smith, Lowell T. Nicholson, Jutamas Suwanpradid, Rachel A. Glenn, Nicole M. Knape, Priya Alagesan, Jaimee N. Gundry, Thomas S. Wehrman, Amber Reck Atwater, Michael D. Gunn, Amanda S. MacLeod, Sudarshan Rajagopal

    The chemokine receptor CXCR3 plays a central role in inflammation by mediating effector/memory T cell migration in various diseases; however, drugs targeting CXCR3 and other chemokine receptors are largely ineffective in treating inflammation. Chemokines, the endogenous peptide ligands of chemokine receptors, can exhibit so-called biased agonism by selectively activating either G protein– or β-arrestin–mediated signaling after receptor binding. Biased agonists might be used as more targeted therapeutics to differentially regulate physiological responses, such as immune cell migration. To test whether CXCR3-mediated physiological responses could be segregated by G protein– and β-arrestin–mediated signaling, we identified and characterized small-molecule biased agonists of the receptor. In a mouse model of T cell–mediated allergic contact hypersensitivity (CHS), topical application of a β-arrestin–biased, but not a G protein–biased, agonist potentiated inflammation. T cell recruitment was increased by the β-arrestin–biased agonist, and biopsies of patients with allergic CHS demonstrated coexpression of CXCR3 and β-arrestin in T cells. In mouse and human T cells, the β-arrestin–biased agonist was the most efficient at stimulating chemotaxis. Analysis of phosphorylated proteins in human lymphocytes showed that β-arrestin–biased signaling activated the kinase Akt, which promoted T cell migration. This study demonstrates that biased agonists of CXCR3 produce distinct physiological effects, suggesting discrete roles for different endogenous CXCR3 ligands and providing evidence that biased signaling can affect the clinical utility of drugs targeting CXCR3 and other chemokine receptors.

    更新日期:2018-11-07
  • Engineering allosteric regulation in protein kinases
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-06
    David Pincus, Jai P. Pandey, Zoë A. Feder, Pau Creixell, Orna Resnekov, Kimberly A. Reynolds

    Phosphoregulation, in which the addition of a negatively charged phosphate group modulates protein activity, enables dynamic cellular responses. To understand how new phosphoregulation might be acquired, we mutationally scanned the surface of a prototypical yeast kinase (Kss1) to identify potential regulatory sites. The data revealed a set of spatially distributed “hotspots” that might have coevolved with the active site and preferentially modulated kinase activity. By engineering simple consensus phosphorylation sites at these hotspots, we rewired cell signaling in yeast. Using the same approach with a homolog yeast mitogen-activated protein kinase, Hog1, we introduced new phosphoregulation that modified its localization and signaling dynamics. Beyond revealing potential use in synthetic biology, our findings suggest that the identified hotspots contribute to the diversity of natural allosteric regulatory mechanisms in the eukaryotic kinome and, given that some are mutated in cancers, understanding these hotspots may have clinical relevance to human disease.

    更新日期:2018-11-07
  • Mammalian pigmentation is regulated by a distinct cAMP-dependent mechanism that controls melanosome pH
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-06
    Dalee Zhou, Koji Ota, Charlee Nardin, Michelle Feldman, Adam Widman, Olivia Wind, Amanda Simon, Michael Reilly, Lonny R. Levin, Jochen Buck, Kazumasa Wakamatsu, Shosuke Ito, Jonathan H. Zippin

    The production of melanin increases skin pigmentation and reduces the risk of skin cancer. Melanin production depends on the pH of melanosomes, which are more acidic in lighter-skinned than in darker-skinned people. We showed that inhibition of soluble adenylyl cyclase (sAC) controlled pigmentation by increasing the pH of melanosomes both in cells and in vivo. Distinct from the canonical melanocortin 1 receptor (MC1R)–dependent cAMP pathway that controls pigmentation by altering gene expression, we found that inhibition of sAC increased pigmentation by increasing the activity of tyrosinase, the rate-limiting enzyme in melanin synthesis, which is more active at basic pH. We demonstrated that the effect of sAC activity on pH and melanin production in human melanocytes depended on the skin color of the donor. Last, we identified sAC inhibitors as a new class of drugs that increase melanosome pH and pigmentation in vivo, suggesting that pharmacologic inhibition of this pathway may affect skin cancer risk or pigmentation conditions.

    更新日期:2018-11-07
  • New connections: Taking advantage of bias
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-06
    John F. Foley

    Biased chemokine receptor agonists and antagonists that target specific signaling pathways may provide therapeutic benefit.

    更新日期:2018-11-07
  • The RAS-ERK pathway: A route for couples
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-30
    Eugenio Santos, Piero Crespo

    Data accumulated over more than three decades demonstrate that the assembly of macrocomplexes, mainly of dimers, is widespread among the members of the different tiers that constitute the RAS-ERK pathway. In this issue of Science Signaling, Yuan et al. report that MEK1 homodimerization is necessary for signal transduction through the RAF-ERK pathway and that cancer-related MEK1 mutations confer enhanced dimerization and resistance to MEK inhibitors. These findings endorse interference with RAS-ERK pathway–component dimerization as a potential therapeutic strategy in cancer patients.

    更新日期:2018-10-31
  • RSK2 contributes to myogenic vasoconstriction of resistance arteries by activating smooth muscle myosin and the Na+/H+ exchanger
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-30
    Mykhaylo V. Artamonov, Swapnil K. Sonkusare, Miranda E. Good, Ko Momotani, Masumi Eto, Brant E. Isakson, Thu H. Le, Eric L. Cope, Zygmunt S. Derewenda, Urszula Derewenda, Avril V. Somlyo

    Smooth muscle contraction is triggered when Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates the regulatory light chain of myosin (RLC20). However, blood vessels from Mlck-deficient mouse embryos retain the ability to contract, suggesting the existence of additional regulatory mechanisms. We showed that the p90 ribosomal S6 kinase 2 (RSK2) also phosphorylated RLC20 to promote smooth muscle contractility. Active, phosphorylated RSK2 was present in mouse resistance arteries under normal basal tone, and phosphorylation of RSK2 increased with myogenic vasoconstriction or agonist stimulation. Resistance arteries from Rsk2-deficient mice were dilated and showed reduced myogenic tone and RLC20 phosphorylation. RSK2 phosphorylated Ser19 in RLC in vitro. In addition, RSK2 phosphorylated an activating site in the Na+/H+ exchanger (NHE-1), resulting in cytosolic alkalinization and an increase in intracellular Ca2+ that promotes vasoconstriction. NHE-1 activity increased upon myogenic constriction, and the increase in intracellular pH was suppressed in Rsk2-deficient mice. In pressured arteries, RSK2-dependent activation of NHE-1 was associated with increased intracellular Ca2+ transients, which would be expected to increase MLCK activity, thereby contributing to basal tone and myogenic responses. Accordingly, Rsk2-deficient mice had lower blood pressure than normal littermates. Thus, RSK2 mediates a procontractile signaling pathway that contributes to the regulation of basal vascular tone, myogenic vasoconstriction, and blood pressure and may be a potential therapeutic target in smooth muscle contractility disorders.

    更新日期:2018-10-31
  • Modulation of Cl− signaling and ion transport by recruitment of kinases and phosphatases mediated by the regulatory protein IRBIT
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-30
    Laura Vachel, Nikolay Shcheynikov, Osamu Yamazaki, Moran Fremder, Ehud Ohana, Aran Son, Dong Min Shin, Ai Yamazaki-Nakazawa, Chin-Rang Yang, Mark A. Knepper, Shmuel Muallem

    IRBIT is a multifunctional protein that controls the activity of various epithelial ion transporters including NBCe1-B. Interaction with IRBIT increases NBCe1-B activity and exposes two cryptic Cl−-sensing GXXXP sites that enable regulation of NBCe1-B by intracellular Cl− (Cl−in). Here, phosphoproteomic analysis revealed that IRBIT controlled five phosphorylation sites in NBCe1-B that determined both the active conformation of the transporter and its regulation by Cl−in. Mutational analysis suggested that the phosphorylation status of Ser232, Ser233, and Ser235 was regulated by IRBIT and determined whether NBCe1 transporters are in active or inactive conformations. The absence of phosphorylation at Ser232, Ser233, or Ser235 produced NBCe1-B in the conformations pSer233/pSer235, pSer232/pSer235, or pSer232/pSer233, respectively. The activity of the pSer233/pSer235 form was similar to that of IRBIT-activated NBCe1-B, but it was insensitive to inhibition by Cl−in. The properties of the pSer232/pSer235 form were similar to those of wild-type NBCe1-B, whereas the pSer232/pSer233 form was partially active, further activated by IRBIT, but retained inhibition by Cl−in. Furthermore, IRBIT recruited the phosphatase PP1 and the kinase SPAK to control phosphorylation of Ser65, which affected Cl−in sensing by the 32GXXXP36 motif. IRBIT also recruited the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of Ser12, which affected Cl−in sensing by the 194GXXXP198 motif. Ser232, Ser233, and Ser235 are conserved in all NBCe1 variants and affect their activity. These findings reveal how multiple kinase and phosphatase pathways use phosphorylation sites to fine-tune a transporter, which have important implications for epithelial fluid and HCO3− secretion.

    更新日期:2018-10-31
  • Activating mutations in MEK1 enhance homodimerization and promote tumorigenesis
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-30
    Jimin Yuan, Wan Hwa Ng, Zizi Tian, Jiajun Yap, Manuela Baccarini, Zhongzhou Chen, Jiancheng Hu

    RAS-RAF-MEK-ERK signaling has a well-defined role in cancer biology. Although aberrant pathway activation occurs mostly upstream of the kinase MEK, mutations in MEK are prevalent in some cancer subsets. Here, we found that cancer-related, activating mutations in MEK can be classified into two groups: those that relieve inhibitory interactions with the helix A region and those that are in-frame deletions of the β3-αC loop, which enhance MEK1 homodimerization. The former, helix A–associated mutants, are inhibited by traditional MEK inhibitors. However, we found that the increased homodimerization associated with the loop-deletion mutants promoted intradimer cross-phosphorylation of the activation loop and conferred differential resistance to MEK inhibitors both in vitro and in vivo. MEK1 dimerization was required both for its activation by the kinase RAF and for its catalytic activity toward the kinase ERK. Our findings not only identify a previously unknown group of MEK mutants and provide insight into some key steps in RAF-MEK-ERK activation but also have implications for the design of therapies targeting RAS-ERK signaling in cancers.

    更新日期:2018-10-31
  • Pathogen-driven vascularization
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-30
    Annalisa M. VanHook

    A cell wall component of mycobacteria stimulates vascularization of granulomas.

    更新日期:2018-10-31
  • A call to arms: Unifying the fight against resistance
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-23
    Alexis Kaushansky, Lizbeth Hedstrom, Aaron Goldman, Juswinder Singh, Priscilla L. Yang, Pradipsinh K. Rathod, Michael Cynamon, Dominik Wodarz, Daruka Mahadevan, Andrew Tomaras, Manuel A. Navia, Celia A. Schiffer

    This Editorial discusses the state of research on drug resistance in the fields of cancer, infectious disease, and agriculture. Reaching across the aisle for a more cross-collaborative approach may lead to exciting breakthroughs toward tackling the challenges of drug resistance in each field.

    更新日期:2018-10-23
  • ER-mitochondria cross-talk is regulated by the Ca2+ sensor NCS1 and is impaired in Wolfram syndrome
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-23
    Claire Angebault, Jérémy Fauconnier, Simone Patergnani, Jennifer Rieusset, Alberto Danese, Corentin A. Affortit, Jolanta Jagodzinska, Camille Mégy, Mélanie Quiles, Chantal Cazevieille, Julia Korchagina, Delphine Bonnet-Wersinger, Dan Milea, Christian Hamel, Paolo Pinton, Marc Thiry, Alain Lacampagne, Benjamin Delprat, Cécile Delettre

    Communication between the endoplasmic reticulum (ER) and mitochondria plays a pivotal role in Ca2+ signaling, energy metabolism, and cell survival. Dysfunction in this cross-talk leads to metabolic and neurodegenerative diseases. Wolfram syndrome is a fatal neurodegenerative disease caused by mutations in the ER-resident protein WFS1. Here, we showed that WFS1 formed a complex with neuronal calcium sensor 1 (NCS1) and inositol 1,4,5-trisphosphate receptor (IP3R) to promote Ca2+ transfer between the ER and mitochondria. In addition, we found that NCS1 abundance was reduced in WFS1-null patient fibroblasts, which showed reduced ER-mitochondria interactions and Ca2+ exchange. Moreover, in WFS1-deficient cells, NCS1 overexpression not only restored ER-mitochondria interactions and Ca2+ transfer but also rescued mitochondrial dysfunction. Our results describe a key role of NCS1 in ER-mitochondria cross-talk, uncover a pathogenic mechanism for Wolfram syndrome, and potentially reveal insights into the pathogenesis of other neurodegenerative diseases.

    更新日期:2018-10-23
  • Sphingosine 1-phosphate stimulates eyelid closure in the developing rat by stimulating EGFR signaling
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-23
    Ganlan Bian, Caiyong Yu, Ling Liu, Chao Fang, Kun Chen, Pan Ren, Qian Zhang, Fangfang Liu, Kun Zhang, Qian Xue, Jie Xiang, Hongmin Guo, Jun Song, Yu Zhao, Wutian Wu, Sookja K. Chung, Ruixia Sun, Gong Ju, Jian Wang

    In many mammals, the eyelids migrate over the eye and fuse during embryogenesis to protect the cornea from damage during birth and early life. Loss-of-function mutations affecting the epidermal growth factor receptor (EGFR) signaling pathway cause an eyes-open-at-birth (EOB) phenotype in rodents. We identified an insertional mutation in Spinster homolog 2 (Spns2) in a strain of transgenic rats exhibiting the EOB phenotype. Spns2, a sphingosine 1-phosphate (S1P) transporter that releases S1P from cells, was enriched at the tip of developing eyelids in wild-type rat embryos. Spns2 expression or treatment with S1P or any one of several EGFR ligands rescued the EOB Spns2 mutant phenotype in vivo and in tissue explants in vitro and rescued the formation of stress fibers in primary keratinocytes from mutants. S1P signaled through the receptors S1PR1, S1PR2, and S1PR3 to activate extracellular signal–regulated kinase (ERK) and EGFR-dependent mitogen-activated protein kinase kinase kinase 1 (MEKK1)–c-Jun signaling. S1P also induced the nuclear translocation of the transcription factor MAL in a manner dependent on EGFR signaling. MAL and c-Jun stimulated the expression of the microRNAs miR-21 and miR-222, both of which target the metalloprotease inhibitor TIMP3, thus promoting metalloprotease activity. The metalloproteases ADAM10 and ADAM17 stimulated EGFR signaling by cleaving a membrane-anchored form of EGF to release the ligand. Our results outline a network by which S1P transactivates EGFR signaling through a complex mechanism involving feedback between several intra- and extracellular molecules to promote eyelid fusion in the developing rat.

    更新日期:2018-10-23
  • The Src family kinase Fgr is a transforming oncoprotein that functions independently of SH3-SH2 domain regulation
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-23
    Kexin Shen, Jamie A. Moroco, Ravi K. Patel, Haibin Shi, John R. Engen, Heather R. Dorman, Thomas E. Smithgall

    Fgr is a member of the Src family of nonreceptor tyrosine kinases, which are overexpressed and constitutively active in many human cancers. Fgr expression is restricted to myeloid hematopoietic cells and is markedly increased in a subset of bone marrow samples from patients with acute myeloid leukemia (AML). Here, we investigated the oncogenic potential of Fgr using Rat-2 fibroblasts that do not express the kinase. Expression of either wild-type or regulatory tail-mutant constructs of Fgr promoted cellular transformation (inferred from colony formation in soft agar), which was accompanied by phosphorylation of the Fgr activation loop, suggesting that the kinase domain of Fgr functions independently of regulation by its noncatalytic SH3-SH2 region. Unlike other family members, recombinant Fgr was not activated by SH3-SH2 domain ligands. However, hydrogen-deuterium exchange mass spectrometry data suggested that the regulatory SH3 and SH2 domains packed against the back of the kinase domain in a Src-like manner. Sequence alignment showed that the activation loop of Fgr was distinct from that of all other Src family members, with proline rather than alanine at the +2 position relative to the activation loop tyrosine. Substitution of the activation loop of Fgr with the sequence from Src partially inhibited kinase activity and suppressed colony formation. Last, Fgr expression enhanced the sensitivity of human myeloid progenitor cells to the cytokine GM-CSF. Because its kinase domain is not sensitive to SH3-SH2–mediated control, simple overexpression of Fgr without mutation may contribute to oncogenic transformation in AML and other blood cancers.

    更新日期:2018-10-23
  • Flipping in a kinase fold for AMPylation
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-23
    Wei Wong

    The pseudokinase SelO mediates AMPylation instead of phosphorylation due to the flipped orientation of ATP in the active site.

    更新日期:2018-10-23
  • Convergence of VEGF and YAP/TAZ signaling: Implications for angiogenesis and cancer biology
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-16
    Ameer L. Elaimy, Arthur M. Mercurio

    Vascular endothelial growth factor (VEGF) stimulates endothelial cells to promote both developmental and pathological angiogenesis. VEGF also directly affects tumor cells and is associated with the initiation, progression, and recurrence of tumors, as well as the emergence and maintenance of cancer stem cells (CSCs). Studies have uncovered the importance of the transcriptional regulators YAP and TAZ in mediating VEGF signaling. For example, VEGF stimulates the GTPase activity of Rho family members and thereby alters cytoskeletal dynamics, which contributes to the activation of YAP and TAZ. In turn, YAP- and TAZ-mediated changes in gene expression sustain Rho family member activity and cytoskeletal effects to promote both vascular growth and remodeling in endothelial cells and the acquisition of stem-like traits in tumor cells. In this Review, we discuss how these findings further explain the pathophysiological roles of VEGF and YAP/TAZ, identify their connections to other receptor-mediated pathways, and reveal ways of therapeutically targeting their convergent signals in patients.

    更新日期:2018-10-17
  • Biased antagonism of CXCR4 avoids antagonist tolerance
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-16
    Ben Hitchinson, Jonathan M. Eby, Xianlong Gao, Francois Guite-Vinet, Joshua J. Ziarek, Hazem Abdelkarim, Youngshim Lee, Yukari Okamoto, Sojin Shikano, Matthias Majetschak, Nikolaus Heveker, Brian F. Volkman, Nadya I. Tarasova, Vadim Gaponenko

    Repeated dosing of drugs targeting G protein–coupled receptors can stimulate antagonist tolerance, which reduces their efficacy; thus, strategies to avoid tolerance are needed. The efficacy of AMD3100, a competitive antagonist of the chemokine receptor CXCR4 that mobilizes leukemic blasts from the bone marrow into the blood to sensitize them to chemotherapy, is reduced after prolonged treatment. Tolerance to AMD3100 increases the abundance of CXCR4 on the surface of leukemic blasts, which promotes their rehoming to the bone marrow. AMD3100 inhibits both G protein signaling by CXCR4 and β-arrestin1/2–dependent receptor endocytosis. We demonstrated that biased antagonists of G protein–dependent chemotaxis but not β-arrestin1/2 recruitment and subsequent receptor endocytosis avoided tolerance. The peptide antagonist X4-2-6, which is derived from transmembrane helix 2 and extracellular loop 1 of CXCR4, limited chemotaxis and signaling but did not promote CXCR4 accumulation on the cell surface or cause tolerance. The activity of X4-2-6 was due to its distinct mechanism of inhibition of CXCR4. The peptide formed a ternary complex with the receptor and its ligand, the chemokine CXCL12. Within this complex, X4-2-6 released the portion of CXCL12 critical for receptor-mediated activation of G proteins but enabled the rest of the chemokine to recruit β-arrestins to the receptor. In contrast, AMD3100 displaced all components of the chemokine responsible for CXCR4 activation. We further identified a small molecule with similar biased antagonist properties to those of X4-2-6, which may provide a viable alternative to patients when antagonist tolerance prevents drugs from reaching efficacy.

    更新日期:2018-10-17
  • Decreased abundance of TRESK two-pore domain potassium channels in sensory neurons underlies the pain associated with bone metastasis
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-16
    Yue Yang, Song Li, Zi-Run Jin, Hong-Bo Jing, Hong-Yan Zhao, Bo-Heng Liu, Ya-Jing Liang, Ling-Yu Liu, Jie Cai, You Wan, Guo-Gang Xing

    Cancer-associated pain is debilitating. Understanding the mechanisms that cause it can inform drug development that may improve quality of life in patients. Here, we found that the reduced abundance of potassium channels called TRESK in dorsal root ganglion (DRG) neurons sensitized nociceptive sensory neurons and cancer-associated pain. Overexpressing TRESK in DRG neurons suppressed tumor-induced neuronal hyperexcitability and pain hypersensitivity in bone metastasis model rats, whereas knocking down TRESK increased neuronal hyperexcitability and pain hypersensitivity in normal rats. Mechanistically, tumor-associated production of vascular endothelial growth factor (VEGF) activated the receptor VEGFR2 on DRGs, which increased the abundance of the calcineurin inhibitor DSCR1, which, in turn, decreased calcineurin-mediated activation of the transcription factor NFAT, thereby reducing the transcription of the gene encoding TRESK. Intrathecal application of exogenous calcineurin to tumor-bearing rats rescued TRESK abundance and abrogated both DRG hyperexcitability and pain hypersensitivity, whereas either inhibition or knockdown of calcineurin in normal rats reduced TRESK abundance and increased DRG excitability and pain sensitivity. These findings identify a potentially targetable mechanism that may cause bone metastasis–associated pain in cancer patients.

    更新日期:2018-10-17
  • G protein subtype–specific signaling bias in a series of CCR5 chemokine analogs
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-16
    Emily Lorenzen, Emilie Ceraudo, Yamina A. Berchiche, Carlos A. Rico, Alexandre Fürstenberg, Thomas P. Sakmar, Thomas Huber

    Chemokines and some chemical analogs of chemokines prevent cellular HIV-1 entry when bound to the HIV-1 coreceptors C-C chemokine receptor 5 (CCR5) or C-X-C chemokine receptor 4 (CXCR4), which are G protein–coupled receptors (GPCRs). The ideal HIV-1 entry blocker targeting the coreceptors would display ligand bias and avoid activating G protein–mediated pathways that lead to inflammation. We compared CCR5-dependent activation of second messenger pathways in a single cell line. We studied two endogenous chemokines [RANTES (also known as CCL5) and MIP-1α (also known as CCL3)] and four chemokine analogs of RANTES (5P12-, 5P14-, 6P4-, and PSC-RANTES). We found that CCR5 signaled through both Gi/o and Gq/11. IP1 accumulation and Ca2+ flux arose from Gq/11 activation, rather than from Gβγ subunit release after Gi/o activation as had been previously proposed. The 6P4- and PSC-RANTES analogs were superagonists for Gq/11 activation, whereas the 5P12- and 5P14-RANTES analogs displayed a signaling bias for Gi/o. These results demonstrate that RANTES analogs elicit G protein subtype–specific signaling bias and can cause CCR5 to couple preferentially to Gq/11 rather than to Gi/o signaling pathways. We propose that G protein subtype–specific signaling bias may be a general feature of GPCRs that can couple to more than one G protein family.

    更新日期:2018-10-17
  • New connections: VEGF beyond the vasculature
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-16
    Leslie K. Ferrarelli

    The growth factor VEGF promotes cancer-associated stem cell biology and pain, as well as angiogenesis.

    更新日期:2018-10-17
  • The protein kinase p38α destabilizes p63 to limit epidermal stem cell frequency and tumorigenic potential
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-09
    Min-Kyung Choo, Stefan Kraft, Caterina Missero, Jin Mo Park

    The molecular circuitry directing tissue development and homeostasis is hardwired by genetic programs but may also be subject to fine-tuning or major modification by environmental conditions. It remains unclear whether such malleability is at work—particularly in tissues directly in contact with the environment—and contributes to their optimal maintenance and resilience. The protein kinase p38α is activated by physiological cues that signal tissue damage and neoplastic transformation. Here, we found that p38α phosphorylated and thereby destabilized p63, a transcription factor essential for epidermal development. Through this regulatory mechanism, p38α limited the frequency of keratinocytes with stem cell properties and tumorigenic potential. Correspondingly, epidermal loss of p38α expression or activity promoted or correlated with carcinogenesis in mouse and human skin, respectively. Genetic mouse models revealed a tumorigenic mechanism from p38α loss through p63-mediated suppression of the matrix metalloprotease MMP13. These findings illustrate a previously uncharacterized epidermal tumor–suppressive mechanism in which stress-activated signaling induces the contraction of stem cell–like keratinocyte pools.

    更新日期:2018-10-10
  • Preassembled GPCR signaling complexes mediate distinct cellular responses to ultralow ligand concentrations
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-09
    Srgjan Civciristov, Andrew M. Ellisdon, Ryan Suderman, Cindy K. Pon, Bronwyn A. Evans, Oded Kleifeld, Steven J. Charlton, William S. Hlavacek, Meritxell Canals, Michelle L. Halls

    G protein–coupled receptors (GPCRs) are the largest class of cell surface signaling proteins, participate in nearly all physiological processes, and are the targets of 30% of marketed drugs. Typically, nanomolar to micromolar concentrations of ligand are used to activate GPCRs in experimental systems. We detected GPCR responses to a wide range of ligand concentrations, from attomolar to millimolar, by measuring GPCR-stimulated production of cyclic adenosine monophosphate (cAMP) with high spatial and temporal resolution. Mathematical modeling showed that femtomolar concentrations of ligand activated, on average, 40% of the cells in a population provided that a cell was activated by one to two binding events. Furthermore, activation of the endogenous β2-adrenergic receptor (β2AR) and muscarinic acetylcholine M3 receptor (M3R) by femtomolar concentrations of ligand in cell lines and human cardiac fibroblasts caused sustained increases in nuclear translocation of extracellular signal–regulated kinase (ERK) and cytosolic protein kinase C (PKC) activity, respectively. These responses were spatially and temporally distinct from those that occurred in response to higher concentrations of ligand and resulted in a distinct cellular proteomic profile. This highly sensitive signaling depended on the GPCRs forming preassembled, higher-order signaling complexes at the plasma membrane. Recognizing that GPCRs respond to ultralow concentrations of neurotransmitters and hormones challenges established paradigms of drug action and provides a previously unappreciated aspect of GPCR activation that is quite distinct from that typically observed with higher ligand concentrations.

    更新日期:2018-10-10
  • IL-17 integrates multiple self-reinforcing, feed-forward mechanisms through the RNA binding protein Arid5a
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-09
    Nilesh Amatya, Erin E. Childs, J. Agustin Cruz, Felix E. Y. Aggor, Abhishek V. Garg, Andrea J. Berman, Johann E. Gudjonsson, Ulus Atasoy, Sarah L. Gaffen

    Interleukin-17A (IL-17A) not only stimulates immunity to fungal pathogens but also contributes to autoimmune pathology. IL-17 is only a modest activator of transcription in experimental tissue culture settings. However, IL-17 controls posttranscriptional events that enhance the expression of target mRNAs. Here, we showed that the RNA binding protein (RBP) Arid5a (AT-rich interactive domain-containing protein 5a) integrated multiple IL-17–driven signaling pathways through posttranscriptional control of mRNA. IL-17 induced expression of Arid5a, which was recruited to the adaptor TRAF2. Arid5a stabilized IL-17–induced cytokine transcripts by binding to their 3′ untranslated regions and also counteracted mRNA degradation mediated by the endoribonuclease MCPIP1 (Regnase-1). Arid5a inducibly associated with the eukaryotic translation initiation complex and facilitated the translation of the transcription factors (TFs) IκBζ (Nfkbiz ) and C/EBPβ (Cebpb). These TFs in turn transactivated IL-17–dependent promoters. Together, these data indicated that Arid5a orchestrates a feed-forward amplification loop, which promoted IL-17 signaling by controlling mRNA stability and translation.

    更新日期:2018-10-10
  • Promoting cytokine production
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-09
    John F. Foley

    The helicase DHX9 is required for production of the cytokine IL-6 in response to Toll-like receptor stimulation.

    更新日期:2018-10-10
  • Arid5a makes the IL-17A/F–responsive pathway less arid
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-09
    Anne Puel, Jean-Laurent Casanova

    The cytokine interleukin-17A/F (IL-17A/F) not only protects the skin and mucosae against Candida albicans infection in mice and humans but also promotes autoimmunity and autoinflammation in mice. In this issue of Science Signaling, Amatya et al. report that the RNA binding protein Arid5a promotes responses to IL-17A/F through multiple mechanisms.

    更新日期:2018-10-10
  • Neratinib is effective in breast tumors bearing both amplification and mutation of ERBB2 (HER2)
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-09
    Emiliano Cocco, F. Javier Carmona, Pedram Razavi, Helen H. Won, Yanyan Cai, Valentina Rossi, Carmen Chan, James Cownie, Joanne Soong, Eneda Toska, Sophie G. Shifman, Ivana Sarotto, Peter Savas, Michael J. Wick, Kyriakos P. Papadopoulos, Alyssa Moriarty, Richard E. Cutler, Francesca Avogadri-Connors, Alshad S. Lalani, Richard P. Bryce, Sarat Chandarlapaty, David M. Hyman, David B. Solit, Valentina Boni, Sherene Loi, José Baselga, Michael F. Berger, Filippo Montemurro, Maurizio Scaltriti

    Mutations in ERBB2, the gene encoding epidermal growth factor receptor (EGFR) family member HER2, are common in and drive the growth of “HER2-negative” (not ERBB2 amplified) tumors but are rare in “HER2-positive” (ERBB2 amplified) breast cancer. We analyzed DNA-sequencing data from HER2-positive patients and used cell lines and a patient-derived xenograft model to test the consequence of HER2 mutations on the efficacy of anti-HER2 agents such as trastuzumab, lapatinib, and neratinib, an irreversible pan-EGFR inhibitor. HER2 mutations were present in ~7% of HER2-positive tumors, all of which were metastatic but not all were previously treated. Compared to HER2 amplification alone, in both patients and cultured cell lines, the co-occurrence of HER2 mutation and amplification was associated with poor response to trastuzumab and lapatinib, the standard-of-care anti-HER2 agents. In mice, xenografts established from a patient whose HER2-positive tumor acquired a D769Y mutation in HER2 after progression on trastuzumab-based therapy were resistant to trastuzumab or lapatinib but were sensitive to neratinib. Clinical data revealed that six heavily pretreated patients with tumors bearing coincident HER2 amplification and mutation subsequently exhibited a statistically significant response to neratinib monotherapy. Thus, these findings indicate that coincident HER2 mutation reduces the efficacy of therapies commonly used to treat HER2-positive breast cancer, particularly in metastatic and previously HER2 inhibitor–treated patients, as well as potentially in patients scheduled for first-line treatment. Therefore, we propose that clinical studies testing the efficacy of neratinib are warranted selectively in breast cancer patients whose tumors carry both amplification and mutation of ERBB2/HER2.

    更新日期:2018-10-10
  • The receptor tyrosine kinase HIR-1 coordinates HIF-independent responses to hypoxia and extracellular matrix injury
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-02
    Roman Vozdek, Yong Long, Dengke K. Ma

    Inadequate tissue oxygen, or hypoxia, is a central concept in the pathophysiology of ischemic disorders and cancer. Hypoxia promotes extracellular matrix (ECM) remodeling, cellular metabolic adaptation, and cancer cell metastasis. To discover new pathways through which cells respond to hypoxia, we performed a large-scale forward genetic screen in Caenorhabditis elegans and identified a previously uncharacterized receptor tyrosine kinase named HIR-1. Loss of function in hir-1 phenocopied the impaired ECM integrity associated with hypoxia or deficiency in the oxygen-dependent dual oxidase, heme peroxidases, or cuticular collagens involved in ECM homeostasis. Genetic suppressor screens identified NHR-49 and MDT-15 as transcriptional regulators downstream of HIR-1. Furthermore, hir-1 mutants showed defects in adapting to and recovering from prolonged severe hypoxia. We propose that C. elegans HIR-1 coordinates hypoxia-inducible factor–independent responses to hypoxia and hypoxia-associated ECM remodeling through mechanisms that are likely conserved in other organisms.

    更新日期:2018-10-03
  • The LKB1–AMPK-α1 signaling pathway triggers hypoxic pulmonary vasoconstriction downstream of mitochondria
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-02
    Javier Moral-Sanz, Sophronia A. Lewis, Sandy MacMillan, Fiona A. Ross, Adrian Thomson, Benoit Viollet, Marc Foretz, Carmel Moran, D. Grahame Hardie, A. Mark Evans

    Hypoxic pulmonary vasoconstriction (HPV), which aids ventilation-perfusion matching in the lungs, is triggered by mechanisms intrinsic to pulmonary arterial smooth muscles. The unique sensitivity of these muscles to hypoxia is conferred by mitochondrial cytochrome c oxidase subunit 4 isoform 2, the inhibition of which has been proposed to trigger HPV through increased generation of mitochondrial reactive oxygen species. Contrary to this model, we have shown that the LKB1–AMPK-α1 signaling pathway is critical to HPV. Spectral Doppler ultrasound revealed that deletion of the AMPK-α1 catalytic subunit blocked HPV in mice during mild (8% O2) and severe (5% O2) hypoxia, whereas AMPK-α2 deletion attenuated HPV only during severe hypoxia. By contrast, neither of these genetic manipulations affected serotonin-induced reductions in pulmonary vascular flow. HPV was also attenuated by reduced expression of LKB1, a kinase that activates AMPK during energy stress, but not after deletion of CaMKK2, a kinase that activates AMPK in response to increases in cytoplasmic Ca2+. Fluorescence imaging of acutely isolated pulmonary arterial myocytes revealed that AMPK-α1 or AMPK-α2 deletion did not affect mitochondrial membrane potential during normoxia or hypoxia. However, deletion of AMPK-α1, but not of AMPK-α2, blocked hypoxia from inhibiting KV1.5, the classical “oxygen-sensing” K+ channel in pulmonary arterial myocytes. We conclude that LKB1–AMPK-α1 signaling pathways downstream of mitochondria are critical for the induction of HPV, in a manner also supported by AMPK-α2 during severe hypoxia.

    更新日期:2018-10-03
  • New connections: Kinase inhibitors as antivirals
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-02
    Erin R. Williams

    Proteomics approaches identify diverse host kinases as potential antiviral therapeutic targets.

    更新日期:2018-10-03
  • Soluble gp130 prevents interleukin-6 and interleukin-11 cluster signaling but not intracellular autocrine responses
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-02
    Larissa Lamertz, Franziska Rummel, Robin Polz, Paul Baran, Selina Hansen, Georg H. Waetzig, Jens M. Moll, Doreen M. Floss, Jürgen Scheller

    Interleukin-6 (IL-6) is a proinflammatory cytokine of the IL-6 family, members of which signal through a complex of a cytokine-specific receptor and the signal-transducing subunit gp130. The interaction of IL-6 with the membrane-bound IL-6 receptor (IL-6R) and gp130 stimulates “classic signaling,” whereas the binding of IL-6 and a soluble version of the IL-6R to gp130 stimulates “trans-signaling.” Alternatively, “cluster signaling” occurs when membrane-bound IL-6:IL-6R complexes on transmitter cells activate gp130 receptors on neighboring receiver cells. The soluble form of gp130 (sgp130) is a selective trans-signaling inhibitor, but it does not affect classic signaling. We demonstrated that the interaction of soluble gp130 with natural and synthetic membrane-bound IL-6:IL-6R complexes inhibited IL-6 cluster signaling. Similarly, IL-11 cluster signaling through the IL-11R to gp130 was also inhibited by soluble gp130. However, autocrine classic and trans-signaling was not inhibited by extracellular inhibitors such as sgp130 or gp130 antibodies. Together, our results suggest that autocrine IL-6 signaling may occur intracellularly.

    更新日期:2018-10-03
  • The bacterial Ras/Rap1 site-specific endopeptidase RRSP cleaves Ras through an atypical mechanism to disrupt Ras-ERK signaling
    Sci. Signal. (IF 6.378) Pub Date : 2018-10-02
    Marco Biancucci, George Minasov, Avik Banerjee, Alfa Herrera, Patrick J. Woida, Matthew B. Kieffer, Lakshman Bindu, Maria Abreu-Blanco, Wayne F. Anderson, Vadim Gaponenko, Andrew G. Stephen, Matthew Holderfield, Karla J. F. Satchell

    The Ras–extracellular signal–regulated kinase pathway is critical for controlling cell proliferation, and its aberrant activation drives the growth of various cancers. Because many pathogens produce toxins that inhibit Ras activity, efforts to develop effective Ras inhibitors to treat cancer could be informed by studies of Ras inhibition by pathogens. Vibrio vulnificus causes fatal infections in a manner that depends on multifunctional autoprocessing repeats-in-toxin, a toxin that releases bacterial effector domains into host cells. One such domain is the Ras/Rap1-specific endopeptidase (RRSP), which site-specifically cleaves the Switch I domain of the small GTPases Ras and Rap1. We solved the crystal structure of RRSP and found that its backbone shares a structural fold with the EreA/ChaN-like superfamily of enzymes. Unlike other proteases in this family, RRSP is not a metalloprotease. Through nuclear magnetic resonance analysis and nucleotide exchange assays, we determined that the processing of KRAS by RRSP did not release any fragments or cause KRAS to dissociate from its bound nucleotide but instead only locally affected its structure. However, this structural alteration of KRAS was sufficient to disable guanine nucleotide exchange factor–mediated nucleotide exchange and prevent KRAS from binding to RAF. Thus, RRSP is a bacterial effector that represents a previously unrecognized class of protease that disconnects Ras from its signaling network while inducing limited structural disturbance in its target.

    更新日期:2018-10-03
  • Substrate binding allosterically relieves autoinhibition of the pseudokinase TRIB1
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-25
    Sam A. Jamieson, Zheng Ruan, Abigail E. Burgess, Jack R. Curry, Hamish D. McMillan, Jodi L. Brewster, Anita K. Dunbier, Alison D. Axtman, Natarajan Kannan, Peter D. Mace

    The Tribbles family of pseudokinases recruits substrates to the ubiquitin ligase COP1 to facilitate ubiquitylation. CCAAT/enhancer-binding protein (C/EBP) family transcription factors are crucial Tribbles substrates in adipocyte and myeloid cell development. We found that the TRIB1 pseudokinase was able to recruit various C/EBP family members and that the binding of C/EBPβ was attenuated by phosphorylation. To explain the mechanism of C/EBP recruitment, we solved the crystal structure of TRIB1 in complex with C/EBPα, which revealed that TRIB1 underwent a substantial conformational change relative to its substrate-free structure and bound C/EBPα in a pseudosubstrate-like manner. Crystallographic analysis and molecular dynamics and subsequent biochemical assays showed that C/EBP binding triggered allosteric changes that link substrate recruitment to COP1 binding. These findings offer a view of pseudokinase regulation with striking parallels to bona fide kinase regulation—by means of the activation loop and αC helix—and raise the possibility of small molecules targeting either the activation “loop-in” or “loop-out” conformations of Tribbles pseudokinases.

    更新日期:2018-09-26
  • Arrestins and G proteins in cellular signaling: The coin has two sides
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-25
    Vsevolod V. Gurevich, Eugenia V. Gurevich

    Several studies have suggested that arrestin-mediated signaling by GPCRs requires G protein activation; however, in this issue of Science Signaling, Luttrell et al. documented arrestin-dependent activation of ERK1/2 by a number of GPCRs. These studies do not contradict each other, but illustrate the complexity of cellular signaling that cannot and should not be reduced to simplistic models.

    更新日期:2018-09-26
  • MerTK signaling in macrophages promotes the synthesis of inflammation resolution mediators by suppressing CaMKII activity
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-25
    Bishuang Cai, Canan Kasikara, Amanda C. Doran, Rajasekhar Ramakrishnan, Raymond B. Birge, Ira Tabas

    Inflammation resolution counterbalances excessive inflammation and restores tissue homeostasis after injury. Failure of resolution contributes to the pathology of numerous chronic inflammatory diseases. Resolution is mediated by endogenous specialized proresolving mediators (SPMs), which are derived from long-chain fatty acids by lipoxygenase (LOX) enzymes. 5-LOX plays a critical role in the biosynthesis of two classes of SPMs: lipoxins and resolvins. Cytoplasmic localization of the nonphosphorylated form of 5-LOX is essential for SPM biosynthesis, whereas nuclear localization of phosphorylated 5-LOX promotes proinflammatory leukotriene production. We previously showed that MerTK, an efferocytosis receptor on macrophages, promotes SPM biosynthesis by increasing the abundance of nonphosphorylated, cytoplasmic 5-LOX. We now show that activation of MerTK in human macrophages led to ERK-mediated expression of the gene encoding sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2), which decreased the cytosolic Ca2+ concentration and suppressed the activity of calcium/calmodulin-dependent protein kinase II (CaMKII). This, in turn, reduced the activities of the mitogen-activated protein kinase (MAPK) p38 and the kinase MK2, resulting in the increased abundance of the nonphosphorylated, cytoplasmic form of 5-LOX and enhanced SPM biosynthesis. In a zymosan-induced peritonitis model, an inflammatory setting in which macrophage MerTK activation promotes resolution, inhibition of ERK activation delayed resolution, which was characterized by an increased number of neutrophils and decreased amounts of SPMs in tissue exudates. These findings contribute to our understanding of how MerTK signaling induces 5-LOX–derived SPM biosynthesis and suggest a therapeutic strategy to boost inflammation resolution in settings where defective resolution promotes disease progression.

    更新日期:2018-09-26
  • New connections: Integrated search for therapies
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-25
    Leslie K. Ferrarelli

    Integrating “omics” analyses with developmental models reveals therapeutic targets for aggressive cancers.

    更新日期:2018-09-26
  • Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-25
    Louis M. Luttrell, Jialu Wang, Bianca Plouffe, Jeffrey S. Smith, Lama Yamani, Suneet Kaur, Pierre-Yves Jean-Charles, Christophe Gauthier, Mi-Hye Lee, Biswaranjan Pani, Jihee Kim, Seungkirl Ahn, Sudarshan Rajagopal, Eric Reiter, Michel Bouvier, Sudha K. Shenoy, Stéphane A. Laporte, Howard A. Rockman, Robert J. Lefkowitz

    G protein–coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. β-Arrestins (βArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete βArr1/2 and G proteins have cast doubt on the role of β-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of βArr1/2 and reconstitution with βArr1/2 in three different parental and CRISPR-derived βArr1/2 knockout HEK293 cell pairs to assess the effect of βArr1/2 deletion on ERK1/2 activation by four Gs-coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for βArr2 or βArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For β2 adrenergic receptors (β2ARs) and β1ARs, βArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V2 and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with βArr1/2. Loss of desensitization and receptor internalization in CRISPR βArr1/2 knockout cells caused β2AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing βArr1/2. These data suggest that βArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from βArr1/2- or G protein–deleted cells to GPCR behavior in native systems.

    更新日期:2018-09-26
  • Covalent inhibitors of EGFR family protein kinases induce degradation of human Tribbles 2 (TRIB2) pseudokinase in cancer cells
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-25
    Daniel M. Foulkes, Dominic P. Byrne, Wayland Yeung, Safal Shrestha, Fiona P. Bailey, Samantha Ferries, Claire E. Eyers, Karen Keeshan, Carrow Wells, David H. Drewry, William J. Zuercher, Natarajan Kannan, Patrick A. Eyers

    A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule–induced protein down-regulation through drug “off-targets” might be relevant for other inhibitors that serendipitously target pseudokinases.

    更新日期:2018-09-26
  • Single-molecule diffusion-based estimation of ligand effects on G protein–coupled receptors
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-18
    Masataka Yanagawa, Michio Hiroshima, Yuichi Togashi, Mitsuhiro Abe, Takahiro Yamashita, Yoshinori Shichida, Masayuki Murata, Masahiro Ueda, Yasushi Sako

    G protein–coupled receptors (GPCRs) are major drug targets. Developing a method to measure the activities of GPCRs is essential for pharmacology and drug screening. However, it is difficult to measure the effects of a drug by monitoring the receptor on the cell surface; thus, changes in the concentrations of downstream signaling molecules, which depend on the signaling pathway selectivity of the receptor, are often used as an index of receptor activity. We show that single-molecule imaging analysis provides an alternative method for assessing the effects of ligands on GPCRs. Using total internal reflection fluorescence microscopy (TIRFM), we monitored the dynamics of the diffusion of metabotropic glutamate receptor 3 (mGluR3), a class C GPCR, under various ligand conditions. Our single-molecule tracking analysis demonstrated that increases and decreases in the average diffusion coefficient of mGluR3 quantitatively reflected the ligand-dependent inactivation and activation of receptors, respectively. Through experiments with inhibitors and dual-color single-molecule imaging analysis, we found that the diffusion of receptor molecules was altered by common physiological events associated with GPCRs, including G protein binding, and receptor accumulation in clathrin-coated pits. We also confirmed that agonist also decreased the average diffusion coefficient for class A and B GPCRs, demonstrating that this parameter is a good index for estimating ligand effects on many GPCRs regardless of their phylogenetic groups, the chemical properties of the ligands, or G protein–coupling selectivity.

    更新日期:2018-09-19
  • Ca2+ concentration–dependent premature death of igfbp5a−/− fish reveals a critical role of IGF signaling in adaptive epithelial growth
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-18
    Chengdong Liu, Yi Xin, Yan Bai, Grant Lewin, Gen He, Kangsen Mai, Cunming Duan

    The phenotype gap is a challenge for genetically dissecting redundant endocrine signaling pathways, such as the six isoforms in the insulin-like growth factor binding protein (IGFBP) family. Although overexpressed IGFBPs can inhibit or potentiate IGF actions or have IGF-independent actions, mutant mice lacking IGFBP-encoding genes do not exhibit major phenotypes. We found that although zebrafish deficient in igfbp5a did not show overt phenotypes when raised in Ca2+-rich solutions, they died prematurely in low Ca2+ conditions. A group of epithelial cells expressing igfbp5a take up Ca2+ and proliferate under low Ca2+ conditions because of activation of IGF signaling. Deletion of igfbp5a blunted low Ca2+ stress–induced IGF signaling and impaired adaptive proliferation. Reintroducing zebrafish Igfbp5a, but not its ligand binding–deficient mutant, restored adaptive proliferation. Similarly, adaptive proliferation was restored in zebrafish lacking igfbp5a by expression of human IGFBP5, but not two cancer-associated IGFBP5 mutants. Knockdown of IGFBP5 in human colon carcinoma cells resulted in reduced IGF-stimulated cell proliferation. These results reveal a conserved mechanism by which a locally expressed Igfbp regulates organismal Ca2+ homeostasis and survival by activating IGF signaling in epithelial cells and promoting their proliferation in Ca2+-deficient states. These findings underscore the importance of physiological context when analyzing loss-of-function phenotypes of endocrine factors.

    更新日期:2018-09-19
  • Going nuclear with stress
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-18
    Wei Wong

    A mitochondrial-derived peptide regulates nuclear gene expression in response to metabolic stress.

    更新日期:2018-09-19
  • The inositol phosphatase SHIP2 enables sustained ERK activation downstream of FGF receptors by recruiting Src kinases
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-18
    Bohumil Fafilek, Lukas Balek, Michaela Kunova Bosakova, Miroslav Varecha, Alexandru Nita, Tomas Gregor, Iva Gudernova, Jitka Krenova, Somadri Ghosh, Martin Piskacek, Lucie Jonatova, Nicole H. Cernohorsky, Jennifer T. Zieba, Michal Kostas, Ellen Margrethe Haugsten, Jørgen Wesche, Christophe Erneux, Lukas Trantirek, Deborah Krakow, Pavel Krejci

    Sustained activation of extracellular signal–regulated kinase (ERK) drives pathologies caused by mutations in fibroblast growth factor receptors (FGFRs). We previously identified the inositol phosphatase SHIP2 (also known as INPPL1) as an FGFR-interacting protein and a target of the tyrosine kinase activities of FGFR1, FGFR3, and FGFR4. We report that loss of SHIP2 converted FGF-mediated sustained ERK activation into a transient signal and rescued cell phenotypes triggered by pathologic FGFR-ERK signaling. Mutant forms of SHIP2 lacking phosphoinositide phosphatase activity still associated with FGFRs and did not prevent FGF-induced sustained ERK activation, demonstrating that the adaptor rather than the catalytic activity of SHIP2 was required. SHIP2 recruited Src family kinases to the FGFRs, which promoted FGFR-mediated phosphorylation and assembly of protein complexes that relayed signaling to ERK. SHIP2 interacted with FGFRs, was phosphorylated by active FGFRs, and promoted FGFR-ERK signaling at the level of phosphorylation of the adaptor FRS2 and recruitment of the tyrosine phosphatase PTPN11. Thus, SHIP2 is an essential component of canonical FGF-FGFR signal transduction and a potential therapeutic target in FGFR-related disorders.

    更新日期:2018-09-19
  • Phosphatidylinositol 4-phosphate is a major source of GPCR-stimulated phosphoinositide production
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-11
    Rafael Gil de Rubio, Richard F. Ransom, Sundeep Malik, David I. Yule, Arun Anantharam, Alan V. Smrcka

    Phospholipase C (PLC) enzymes hydrolyze the plasma membrane (PM) lipid phosphatidylinositol 4,5-bisphosphate (PI4,5P2) to generate the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG) in response to receptor activation in almost all mammalian cells. We previously found that stimulation of G protein–coupled receptors (GPCRs) in cardiac cells leads to the PLC-dependent hydrolysis of phosphatidylinositol 4-phosphate (PI4P) at the Golgi, a process required for the activation of nuclear protein kinase D (PKD) during cardiac hypertrophy. We hypothesized that GPCR-stimulated PLC activation leading to direct PI4P hydrolysis may be a general mechanism for DAG production. We measured GPCR activation–dependent changes in PM and Golgi PI4P pools in various cells using GFP-based detection of PI4P. Stimulation with various agonists caused a time-dependent reduction in PI4P-associated, but not PI4,5P2-associated, fluorescence at the Golgi and PM. Targeted depletion of PI4,5P2 from the PM before GPCR stimulation had no effect on the depletion of PM or Golgi PI4P, total inositol phosphate (IP) production, or PKD activation. In contrast, acute depletion of PI4P specifically at the PM completely blocked the GPCR-dependent production of IPs and activation of PKD but did not change the abundance of PI4,5P2. Acute depletion of Golgi PI4P had no effect on these processes. These data suggest that most of the PM PI4,5P2 pool is not involved in GPCR-stimulated phosphoinositide hydrolysis and that PI4P at the PM is responsible for the bulk of receptor-stimulated phosphoinositide hydrolysis and DAG production.

    更新日期:2018-09-12
  • The Hippo pathway effector TAZ induces TEAD-dependent liver inflammation and tumors
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-11
    Thijs J. Hagenbeek, Joshua D. Webster, Noelyn M. Kljavin, Matthew T. Chang, Trang Pham, Ho-June Lee, Christiaan Klijn, Allen G. Cai, Klara Totpal, Buvana Ravishankar, Naiying Yang, Da-Hye Lee, Kevin B. Walsh, Georgia Hatzivassiliou, Cecile C. de la Cruz, Stephen E. Gould, Xiumin Wu, Wyne P. Lee, Shuqun Yang, Zhixiang Zhang, Qingyang Gu, Qunsheng Ji, Erica L. Jackson, Dae-Sik Lim, Anwesha Dey

    The Hippo signaling pathway regulates organ size and plays critical roles in maintaining tissue growth, homeostasis, and regeneration. Dysregulated in a wide spectrum of cancers, in mammals, this pathway is regulated by two key effectors, YAP and TAZ, that may functionally overlap. We found that TAZ promoted liver inflammation and tumor development. The expression of TAZ, but not YAP, in human liver tumors positively correlated with the expression of proinflammatory cytokines. Hyperactivated TAZ induced substantial myeloid cell infiltration into the liver and the secretion of proinflammatory cytokines through a TEAD-dependent mechanism. Furthermore, tumors with hyperactivated YAP and TAZ had distinct transcriptional signatures, which included the increased expression of inflammatory cytokines in TAZ-driven tumors. Our study elucidated a previously uncharacterized link between TAZ activity and inflammatory responses that influence tumor development in the liver.

    更新日期:2018-09-12
  • HSV-1 deamidates cGAS
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-11
    Annalisa M. VanHook

    An HSV-1 enzyme represses innate immune responses by deamidating the cytosolic DNA sensor cGAS.

    更新日期:2018-09-12
  • Should the clinic matter to nonphysician scientists?
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-11
    Pierre Jean Le Reste, Eric Chevet

    Translational medicine has been nurtured by the creation of M.D.-Ph.D. programs, a system that now needs to reinvent itself. Now, clinically oriented training programs targeting nonphysician scientists have opened new avenues to improve transdisciplinary approaches in health sciences.

    更新日期:2018-09-12
  • Developmental phosphoproteomics identifies the kinase CK2 as a driver of Hedgehog signaling and a therapeutic target in medulloblastoma
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-11
    Teresa Purzner, James Purzner, Taylor Buckstaff, Giorgio Cozza, Sharareh Gholamin, Jessica M. Rusert, Tom A. Hartl, John Sanders, Nicholas Conley, Xuecai Ge, Marc Langan, Vijay Ramaswamy, Lauren Ellis, Ulrike Litzenburger, Sara Bolin, Johanna Theruvath, Ryan Nitta, Lin Qi, Xiao-Nan Li, Gordon Li, Michael D. Taylor, Robert J. Wechsler-Reya, Lorenzo A. Pinna, Yoon-Jae Cho, Margaret T. Fuller, Joshua E. Elias, Matthew P. Scott

    A major limitation of targeted cancer therapy is the rapid emergence of drug resistance, which often arises through mutations at or downstream of the drug target or through intrinsic resistance of subpopulations of tumor cells. Medulloblastoma (MB), the most common pediatric brain tumor, is no exception, and MBs that are driven by sonic hedgehog (SHH) signaling are particularly aggressive and drug-resistant. To find new drug targets and therapeutics for MB that may be less susceptible to common resistance mechanisms, we used a developmental phosphoproteomics approach in murine granule neuron precursors (GNPs), the developmental cell of origin of MB. The protein kinase CK2 emerged as a driver of hundreds of phosphorylation events during the proliferative, MB-like stage of GNP growth, including the phosphorylation of three of the eight proteins commonly amplified in MB. CK2 was critical to the stabilization and activity of the transcription factor GLI2, a late downstream effector in SHH signaling. CK2 inhibitors decreased the viability of primary SHH-type MB patient cells in culture and blocked the growth of murine MB tumors that were resistant to currently available Hh inhibitors, thereby extending the survival of tumor-bearing mice. Because of structural interactions, one CK2 inhibitor (CX-4945) inhibited both wild-type and mutant CK2, indicating that this drug may avoid at least one common mode of acquired resistance. These findings suggest that CK2 inhibitors may be effective for treating patients with MB and show how phosphoproteomics may be used to gain insight into developmental biology and pathology.

    更新日期:2018-09-12
  • The kinases HipA and HipA7 phosphorylate different substrate pools in Escherichia coli to promote multidrug tolerance
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-11
    Maja Semanjski, Elsa Germain, Katrin Bratl, Andreas Kiessling, Kenn Gerdes, Boris Macek

    The bacterial serine-threonine protein kinase HipA promotes multidrug tolerance by phosphorylating the glutamate-tRNA ligase (GltX), leading to a halt in translation, inhibition of growth, and induction of a physiologically dormant state (persistence). The HipA variant HipA7 substantially increases persistence despite being less efficient at inhibiting cell growth. We postulated that this phenotypic difference was caused by differences in the substrates targeted by both kinases. We overproduced HipA and HipA7 in Escherichia coli and identified their endogenous substrates by SILAC-based quantitative phosphoproteomics. We confirmed that GltX was the main substrate of both kinase variants and likely the primary determinant of persistence. When HipA and HipA7 were moderately overproduced from plasmids, HipA7 targeted only GltX, but HipA phosphorylated several additional substrates involved in translation, transcription, and replication, such as ribosomal protein L11 (RplK) and the negative modulator of replication initiation, SeqA. HipA7 showed reduced kinase activity compared to HipA and targeted a substrate pool similar to that of HipA only when produced from a high–copy number plasmid. The kinase variants also differed in autophosphorylation, which was substantially reduced for HipA7. When produced endogenously from the chromosome, HipA showed no activity because of inhibition by the antitoxin HipB, whereas HipA7 phosphorylated GltX and phage shock protein PspA. Initial testing did not reveal a connection between HipA-induced phosphorylation of RplK and persistence or growth inhibition, suggesting that other HipA-specific substrates were likely responsible for growth inhibition. Our results contribute to the understanding of HipA7 action and present a resource for elucidating HipA-related persistence.

    更新日期:2018-09-12
  • Coping with stress by regulating tRNAs
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-04
    Sebastian Pechmann

    Stress conditions curtail the energetically costly process of messenger RNA translation. In this issue of Science Signaling, Torrent et al. report key evidence for a direct link between codon usage and translation regulation in response to stress.

    更新日期:2018-09-05
  • The pseudokinase MLKL activates PAD4-dependent NET formation in necroptotic neutrophils
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-04
    Akshay A. D’Cruz, Mary Speir, Meghan Bliss-Moreau, Sylvia Dietrich, Shu Wang, Alyce A. Chen, Mathilde Gavillet, Arshed Al-Obeidi, Kate E. Lawlor, James E. Vince, Michelle A. Kelliher, Razq Hakem, Manolis Pasparakis, David A. Williams, Maria Ericsson, Ben A. Croker

    Neutrophil extracellular trap (NET) formation can generate short-term, functional anucleate cytoplasts and trigger loss of cell viability. We demonstrated that the necroptotic cell death effector mixed lineage kinase domain–like (MLKL) translocated from the cytoplasm to the plasma membrane and stimulated downstream NADPH oxidase–independent ROS production, loss of cytoplasmic granules, breakdown of the nuclear membrane, chromatin decondensation, histone hypercitrullination, and extrusion of bacteriostatic NETs. This process was coordinated by receptor-interacting protein kinase-1 (RIPK1), which activated the caspase-8–dependent apoptotic or RIPK3/MLKL-dependent necroptotic death of mouse and human neutrophils. Genetic deficiency of RIPK3 and MLKL prevented NET formation but did not prevent cell death, which was because of residual caspase-8–dependent activity. Peptidylarginine deiminase 4 (PAD4) was activated downstream of RIPK1/RIPK3/MLKL and was required for maximal histone hypercitrullination and NET extrusion. This work defines a distinct signaling network that activates PAD4-dependent NET release for the control of methicillin-resistant Staphylococcus aureus (MRSA) infection.

    更新日期:2018-09-05
  • Glucocorticoids and PD-1
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-04
    John F. Foley

    Signaling by the glucocorticoid receptor in NK cells induces expression of the checkpoint inhibitor PD-1 to prevent immunopathology.

    更新日期:2018-09-05
  • Cells alter their tRNA abundance to selectively regulate protein synthesis during stress conditions
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-04
    Marc Torrent, Guilhem Chalancon, Natalia S. de Groot, Arthur Wuster, M. Madan Babu

    Decoding the information in mRNA during protein synthesis relies on tRNA adaptors, the abundance of which can affect the decoding rate and translation efficiency. To determine whether cells alter tRNA abundance to selectively regulate protein expression, we quantified changes in the abundance of individual tRNAs at different time points in response to diverse stress conditions in Saccharomyces cerevisiae. We found that the tRNA pool was dynamic and rearranged in a manner that facilitated selective translation of stress-related transcripts. Through genomic analysis of multiple data sets, stochastic simulations, and experiments with designed sequences of proteins with identical amino acids but altered codon usage, we showed that changes in tRNA abundance affected protein expression independently of factors such as mRNA abundance. We suggest that cells alter their tRNA abundance to selectively affect the translation rates of specific transcripts to increase the amounts of required proteins under diverse stress conditions.

    更新日期:2018-09-05
  • Targeting nucleotide exchange to inhibit constitutively active G protein α subunits in cancer cells
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-04
    Michael D. Onken, Carol M. Makepeace, Kevin M. Kaltenbronn, Stanley M. Kanai, Tyson D. Todd, Shiqi Wang, Thomas J. Broekelmann, Prabakar Kumar Rao, John A. Cooper, Kendall J. Blumer

    Constitutively active G protein α subunits cause cancer, cholera, Sturge-Weber syndrome, and other disorders. Therapeutic intervention by targeted inhibition of constitutively active Gα subunits in these disorders has yet to be achieved. We found that constitutively active Gαq in uveal melanoma (UM) cells was inhibited by the cyclic depsipeptide FR900359 (FR). FR allosterically inhibited guanosine diphosphate–for–guanosine triphosphate (GDP/GTP) exchange to trap constitutively active Gαq in inactive, GDP-bound Gαβγ heterotrimers. Allosteric inhibition of other Gα subunits was achieved by the introduction of an FR-binding site. In UM cells driven by constitutively active Gαq, FR inhibited second messenger signaling, arrested cell proliferation, reinstated melanocytic differentiation, and stimulated apoptosis. In contrast, FR had no effect on BRAF-driven UM cells. FR promoted UM cell differentiation by reactivating polycomb repressive complex 2 (PRC2)–mediated gene silencing, a heretofore unrecognized effector system of constitutively active Gαq in UM. Constitutively active Gαq and PRC2 therefore provide therapeutic targets for UM. The development of FR analogs specific for other Gα subunit subtypes may provide novel therapeutic approaches for diseases driven by constitutively active Gα subunits or multiple G protein–coupled receptors (GPCRs) where targeting a single receptor is ineffective.

    更新日期:2018-09-05
  • Oncogenic RAS isoforms show a hierarchical requirement for the guanine nucleotide exchange factor SOS2 to mediate cell transformation
    Sci. Signal. (IF 6.378) Pub Date : 2018-09-04
    Erin Sheffels, Nancy E. Sealover, Chenyue Wang, Do Hyung Kim, Isabella A. Vazirani, Elizabeth Lee, Elizabeth M. Terrell, Deborah K. Morrison, Ji Luo, Robert L. Kortum

    About a third of tumors have activating mutations in HRAS, NRAS, or KRAS, genes encoding guanosine triphosphatases (GTPases) of the RAS family. In these tumors, wild-type RAS cooperates with mutant RAS to promote downstream effector activation and cell proliferation and transformation, suggesting that upstream activators of wild-type RAS are important modulators of mutant RAS-driven oncogenesis. The guanine nucleotide exchange factor (GEF) SOS1 mediates KRAS-driven proliferation, but little is understood about the role of SOS2. We found that RAS family members have a hierarchical requirement for the expression and activity of SOS2 to drive cellular transformation. In mouse embryonic fibroblasts (MEFs), SOS2 critically mediated mutant KRAS-driven, but not HRAS-driven, transformation. Sos2 deletion reduced epidermal growth factor (EGF)–dependent activation of wild-type HRAS and phosphorylation of the kinase AKT in cells expressing mutant RAS isoforms. Assays using pharmacological inhibitors revealed a hierarchical requirement for signaling by phosphoinositide 3-kinase (PI3K) in promoting RAS-driven cellular transformation that mirrored the requirement for SOS2. KRAS-driven transformation required the GEF activity of SOS2 and was restored in Sos2−/− MEFs by expression of constitutively activated PI3K. Finally, CRISPR/Cas9-mediated deletion of SOS2 reduced EGF-stimulated AKT phosphorylation and synergized with MEK inhibition to revert the transformed phenotype of human KRAS mutant pancreatic and lung tumor cells. These results indicate that SOS2-dependent PI3K signaling mediates mutant KRAS-driven transformation, revealing therapeutic targets in KRAS-driven cancers. Our data also reveal the importance of three-dimensional culture systems in investigating the mediators of mutant KRAS.

    更新日期:2018-09-05
  • Making cells move with IRE1α
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-28
    Wei Wong

    IRE1α promotes cell migration independently of its function as a transducer of ER stress responses.

    更新日期:2018-08-29
  • TGF-β–mediated enhancement of TH17 cell generation is inhibited by bone morphogenetic protein receptor 1α signaling
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-28
    Lauren M. Browning, Maciej Pietrzak, Michal Kuczma, Colin P. Simms, Agnieszka Kurczewska, Justin M. Refugia, Dustin J. Lowery, Grzegorz Rempala, Dmitriy Gutkin, Leszek Ignatowicz, Pawel Muranski, Piotr Kraj

    The cytokines of the transforming growth factor–β (TGF-β) family promote the growth and differentiation of multiple tissues, but the role of only the founding member, TGF-β, in regulating the immune responses has been extensively studied. TGF-β is critical to prevent the spontaneous activation of self-reactive T cells and sustain immune homeostasis. In contrast, in the presence of proinflammatory cytokines, TGF-β promotes the differentiation of effector T helper 17 (TH17) cells. Abrogating TGF-β receptor signaling prevents the development of interleukin-17 (IL-17)–secreting cells and protects mice from TH17 cell–mediated autoimmunity. We found that the receptor of another member of TGF-β family, bone morphogenetic protein receptor 1α (BMPR1α), regulates T helper cell activation. We found that the differentiation of TH17 cells from naive CD4+ T cells was inhibited in the presence of BMPs. Abrogation of BMPR1α signaling during CD4+ T cell activation induced a developmental program that led to the generation of inflammatory effector cells expressing large amounts of IL-17, IFN-γ, and TNF family cytokines and transcription factors defining the TH17 cell lineage. We found that TGF-β and BMPs cooperated to establish effector cell functions and the cytokine profile of activated CD4+ T cells. Together, our data provide insight into the immunoregulatory function of BMPs.

    更新日期:2018-08-29
  • Selective inhibition of CaV3.2 channels reverses hyperexcitability of peripheral nociceptors and alleviates postsurgical pain
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-28
    Sonja L. Joksimovic, Srdjan M. Joksimovic, Vesna Tesic, Agustin García-Caballero, Simon Feseha, Gerald W. Zamponi, Vesna Jevtovic-Todorovic, Slobodan M. Todorovic

    Pain-sensing sensory neurons of the dorsal root ganglion (DRG) can become sensitized or hyperexcitable in response to surgically induced peripheral tissue injury. We investigated the potential role and molecular mechanisms of nociceptive ion channel dysregulation in acute pain conditions such as those resulting from skin and soft tissue incision. We used selective pharmacology, electrophysiology, and mouse genetics to link increased current densities arising from the CaV3.2 isoform of T-type calcium channels (T-channels) to nociceptive sensitization using a clinically relevant rodent model of skin and deep tissue incision. Furthermore, knockdown of the CaV3.2-targeting deubiquitinating enzyme USP5 or disruption of USP5 binding to CaV3.2 channels in peripheral nociceptors resulted in a robust antihyperalgesic effect in vivo and substantial T-current reduction in vitro. Our study provides mechanistic insight into the role of plasticity in CaV3.2 channel activity after surgical incision and identifies potential targets for perioperative pain that may greatly decrease the need for narcotics and potential for drug abuse.

    更新日期:2018-08-29
Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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