Extracellular Signal-regulated Kinase Activation In Response Research Articles

Effects of a Tripeptide on Mitogen-Activated Protein Kinase and Glycogen Synthase Kinase Activation in a Cell Line Derived from the Foetal Hippocampus of a Trisomy 16 Mouse: an Animal Model of Down Syndrome.

Down syndrome (DS) is a developmental disorder that results from the trisomy of chromosome 21. DS patients show several abnormalities including cognitive deficits. Here, we show enhanced activation of the extracellular signal-regulated kinase (ERK), a kinase that critically regulates synaptic plasticity and memory, in a hippocampal cell line derived from trisomy 16 mouse foetus. In addition, these cells show enhanced activation of p38 mitogen-activated protein kinase (p38 MAPK). The hyper-activation of ERK and p38 MAPK is significantly reduced by a small peptide, Gly-Pro-Glu (GPE), derived from insulin-like growth factor-1. In addition, the trisomic cells show reduced level of inhibitory phosphorylation of glycogen synthase kinase-3β (GSK-3β), which is enhanced by GPE. Furthermore, the trisomic cells do not show ERK activation in response to KCl depolarization or forskolin treatment. Importantly, ERK activation by these stimuli is observed after GPE treatment of the cells. These results suggest that GPE may help reduce aberrant signalling in the trisomic neurons by affecting MAPK and GSK-3β activation.

The Role of PAR2 in TGF-β1-Induced ERK Activation and Cell Motility.

Background: Recently, the expression of proteinase-activated receptor 2 (PAR2) has been shown to be essential for activin receptor-like kinase 5 (ALK5)/SMAD-mediated signaling and cell migration by transforming growth factor (TGF)-β1. However, it is not known whether activation of non-SMAD TGF-β signaling (e.g., RAS–RAF–MEK–extracellular signal-regulated kinase (ERK) signaling) is required for cell migration and whether it is also dependent on PAR2. Methods: RNA interference was used to deplete cells of PAR2, followed by xCELLigence technology to measure cell migration, phospho-immunoblotting to assess ERK1/2 activation, and co-immunoprecipitation to detect a PAR2–ALK5 physical interaction. Results: Inhibition of ERK signaling with the MEK inhibitor U0126 blunted the ability of TGF-β1 to induce migration in pancreatic cancer Panc1 cells. ERK activation in response to PAR2 agonistic peptide (PAR2–AP) was strong and rapid, while it was moderate and delayed in response to TGF-β1. Basal and TGF-β1-dependent ERK, but not SMAD activation, was blocked by U0126 in Panc1 and other cell types indicating that ERK activation is downstream or independent of SMAD signaling. Moreover, cellular depletion of PAR2 in HaCaT cells strongly inhibited TGF-β1-induced ERK activation, while the biased PAR2 agonist GB88 at 10 and 100 µM potentiated TGF-β1-dependent ERK activation and cell migration. Finally, we provide evidence for a physical interaction between PAR2 and ALK5. Our data show that both PAR2–AP- and TGF-β1-induced cell migration depend on ERK activation, that PAR2 expression is crucial for TGF-β1-induced ERK activation, and that the functional cooperation of PAR2 and TGF-β1 involves a physical interaction between PAR2 and ALK5.

YAP and ERK mediated mechanical strain-induced cell cycle progression through RhoA and cytoskeletal dynamics in rat growth plate chondrocytes.

Yes-associated protein (YAP) and extracellular signal-regulated kinase (ERK) have been considered as key regulators in tissue homeostasis, organ development, and tumor formation. However, the roles of YAP and ERK in the mediating strain mechanosensing in the growth plate cartilage have not been determined. In this study, chondrocytes obtained from the growth plate cartilage of 2-week-old Sprague-Dawley rats were subjected to the mechanical strain with different magnitudes and durations at a frequency of 0.5 Hz. We found that YAP and ERK activation in response to mechanical strain was time and magnitude dependent. Pretreatment with a RhoA inhibitor (C3 toxin) or a microfilament cytoskeleton disrupting reagent (cytochalasin D) could suppress their activation. In addition, activated YAP and ERK were able to induce cell cycle progression by up-regulating the expression of cell cycle-related genes. These results shed new light on the function of YAP and ERK in mechanical strain-promoted growth plate development. Our results also provided evidence that RhoA and cytoskeletal dynamics are required for this mechanotransduction. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1121-1129, 2016.

Convergence of dopamine and glutamate signaling onto striatal ERK activation in response to drugs of abuse

Despite their distinct targets, all addictive drugs commonly abused by humans evoke increases in dopamine (DA) concentration within the striatum. The main DA Guanine nucleotide binding protein couple receptors (GPCRs) expressed by medium-sized spiny neurons of the striatum are the D1R and D2R, which are positively and negatively coupled to cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling, respectively. These two DA GPCRs are largely segregated into distinct neuronal populations, where they are co-expressed with glutamate receptors in dendritic spines. Direct and indirect interactions between DA GPCRs and glutamate receptors are the molecular basis by which DA modulates glutamate transmission and controls striatal plasticity and behavior induced by drugs of abuse. A major downstream target of striatal D1R is the extracellular signal-regulated kinase (ERK) kinase pathway. ERK activation by drugs of abuse behaves as a key integrator of D1R and glutamate NMDAR signaling. Once activated, ERK can trigger chromatin remodeling and induce gene expression that permits long-term cellular alterations and drug-induced morphological and behavioral changes. Besides the classical cAMP/PKA pathway, downstream of D1R, recent evidence implicates a cAMP-independent crosstalk mechanism by which the D1R potentiates NMDAR-mediated calcium influx and ERK activation. The mounting evidence of reciprocal modulation of DA and glutamate receptors adds further intricacy to striatal synaptic signaling and is liable to prove relevant for addictive drug-induced signaling, plasticity, and behavior. Herein, we review the evidence that built our understanding of the consequences of this synergistic signaling for the actions of drugs of abuse.

Juvenile ethanol exposure increases rewarding properties of cocaine and morphine in adult DBA/2J mice

Convergent data showed that ethanol exposure during adolescence can alter durably ethanol-related behaviour at adulthood. However, the consequences of juvenile ethanol exposure on the reinforcing effects of other drugs of abuse remain unclear. In the present work, we evaluated in adult male DBA/2J mice the effects of early ethanol exposure on the sensitivity to the incentive effects of cocaine and morphine, and on extracellular signal-regulated kinase (ERK) activation in response to cocaine. Juvenile male mice received intragastric administration of ethanol (2×2.5g/kg/day) or water for 5 days starting on postnatal day 28. When reaching adult age (10 week-old), animals were subjected to an unbiased procedure to assess conditioned place preference (CPP) to cocaine or morphine. In addition, activation of ERK in response to an acute injection of cocaine was investigated using immunoblotting in the striatum and the nucleus accumbens. Mice that have been subjected to early ethanol exposure developed CPP to doses of cocaine (5mg/kg) or morphine (10mg/kg) below the threshold doses to induce CPP in water pre-exposed mice. In addition, early ethanol administration significantly increased striatal ERK phosphorylation normally induced by acute cocaine (10 and 20mg/kg) in adult mice. These results show that, in DBA/2J mice, early exposure to ethanol enhanced the perception of the incentive effects of cocaine and morphine. Ethanol pre-exposure also induced a positive modulation of striatal ERK signalling, in line with the inference that juvenile ethanol intake may contribute to the development of addictive behaviour at adult age.

Estrogens from the Outside In: Alkylphenols, BPA Disrupt ERK Signaling in Vitro

Vol. 119, No. 1 NewsOpen AccessEstrogens from the Outside In: Alkylphenols, BPA Disrupt ERK Signaling in Vitro Julia R. Barrett Julia R. Barrett Search for more papers by this author Published:1 January 2011https://doi.org/10.1289/ehp.119-a34bCited by:1AboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit The body produces estrogens—including estrone (E1), estradiol (E2), and estriol (E3)—that direct reproductive system processes and contribute to the normal function of tissues including the brain, bone, and cardiovascular system. Certain xenoestrogens (estrogenic compounds introduced from outside the body) are suspected of disrupting these activities. In a new study, xenoestrogenic alkylphenols and bisphenol A (BPA) interfered with normal estrogenic signaling in vitro, which suggests they could disrupt normal physiologic function at critical life stages [EHP119(1):104–112; Jeng and Watson].Different estrogen receptors control different functions: receptors in the cell nucleus direct gene transcription, whereas receptors in the cell membrane direct signaling pathways via extracellular signal–regulated kinases (ERKs). ERK-controlled pathways respond to many biochemical stimuli and integrate these signals to direct a cell toward division, differentiation, death, or malignant transformation. The structurally related alkylphenols and BPA interact weakly with nuclear estrogen receptors, but they can have pronounced effects on signaling pathways mediated by estrogen receptors in the cell membrane.In the current study, a rat pituitary cancer cell line was used to study the effect of alkylphenols and BPA on ERK1 and ERK2 activation (measured as phosphorylation), both alone and in combination with each physiologic estrogen. After treatment with each physiologic and environmental estrogen, the researchers measured time-dependent surges in ERK activation. In most cases, E1 and E2 prompted early, intermediate, and late surges in ERK activation at 5, 10–30, and >30 min, respectively; alkylphenols and E3 typically triggered early and late surges. Interestingly, a very low concentration of BPA (10−14 M) yielded a similar two-peak response, but a higher concentration (1 nM) induced a three-peak response like that of E1 and E2. Both BPA concentrations were typical of environmental exposures and, along with ineffective midrange doses, also illustrated the nonmonotonic dose–response relationship characteristic of many estrogenic compounds.When physiologic estrogens and xenoestrogens were combined, the response pattern generally shifted to a single major peak at an intermediate time. Xenoestrogens that caused a strong response when administered alone at a particular point in time or concentration tended to inhibit ERK activation in response to a physiologic estrogen. But at other times or concentrations, the same xenoestrogen might cause a weak response on its own, in which case it would tend to enhance ERK phosphorylation in response to physiologic estrogens.There were exceptions to these general patterns, however, which highlights the need to study effects of individual xenoestrogens at different points in time, at varying concentrations, and in different tissues. The effect of shifts in the patterns of ERK activation are only just beginning to be explored, although it is known that these patterns constitute an important component of information flow within a cell. The correct flow of information is likely to be especially critical during windows of vulnerability that are based in part on life stage.FiguresReferencesRelatedDetailsCited by Park M, Hwang K and Choi K (2011) Diverse animal models to examine potential role(s) and mechanism of endocrine disrupting chemicals on the tumor progression and prevention: Do they have tumorigenic or anti-tumorigenic property?, Laboratory Animal Research, 10.5625/lar.2011.27.4.265, 27:4, (265), . Vol. 119, No. 1 January 2011Metrics About Article Metrics Publication History Originally published1 January 2011Published in print1 January 2011 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days.

Abstract 5358: Epstein-Barr Virus-encoded BARF1 gene stimulates gastric epithelial cell MMP-1 secretion through ERK activation

Abstract The Epstein-Barr virus (EBV), originally isolated from Burkitt's lymphoma tissue, has now been associated with various epithelioid diseases and many other malignancies, including gastric adenocarcinoma. Our interest has been focused on the BARF1 early gene from EBV, which has ability to induce oncogenic transformation in rodent fibroblasts and to immortalize monkey kidney primary epitherial cells. The aim of this study was to investigate the effect of BARF1 expression in gastric cancer cells. First, a pEGFP vector containing the full length BARF1 gene was constructed and transfected into EBV negative gastric cancer cell lines (AGS and SNU601). Microarray analysis was then performed and gene expression profiles were analyzed and compared between the cell containing full length BARF1 gene and the vector control. We found that matrix metalloproteinase-1 (MMP-1) was significantly up-regulated in the cell containing BARF1 gene. The MMP-1 mRNA and protein expression levels are confirmed by RT-PCR and Western blotting. Additionally, the mobility of BARF1 transfectants was increased in scrape-wound migration assay compared with the vector control cells. Because MMP-1 secretion in other cell types is extracellular signal regulated kinase (ERK)-dependent, we examined mitogen-activated protein kinase (MAPK) ERK activation in response to BARF1. BARF1 also induced activation of ERK. To determine whether BARF1-induced MMP-1 secretion was ERK-dependent, we used PD98059 and UO126, the specific inhibitors of MEK, the proximal activator of ERK. UO126 and PD98059 inhibited ERK activation and MMP-1 secretion, indicating that MMP-1 secretion in response to BARF1 was MEK- and ERK-dependent. Because p38 activity mediates MMP-1 secretion in dermal fibroblasts and several other cell types, but inhibits MMP-1 secretion in cytokine-stimulated AGS cells, we also examined the effects of p38 inhibition on MMP-1 secretion in response to BARF1. p38 inhibition with the specific p38 inhibitors, SB203580 and SB202190, enhanced MMP-1 secretion, as well as ERK activation. These results indicate that EBV-encoded BARF1 gene stimulates gastric epithelial cell MMP-1 secretion via ERK activation. This is the first report that a viral protein, BARF1, is capable of regulating MMP-1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5358.

Leukotriene E4 Activates Peroxisome Proliferator-activated Receptor γ and Induces Prostaglandin D2 Generation by Human Mast Cells

Cysteinyl leukotrienes (cys-LTs) are potent inflammatory lipid mediators, of which leukotriene (LT) E(4) is the most stable and abundant in vivo. Although only a weak agonist of established G protein-coupled receptors (GPCRs) for cys-LTs, LTE(4) potentiates airway hyper-responsiveness (AHR) by a cyclooxygenase (COX)-dependent mechanism and induces bronchial eosinophilia. We now report that LTE(4) activates human mast cells (MCs) by a pathway involving cooperation between an MK571-sensitive GPCR and peroxisome proliferator-activated receptor (PPAR)gamma, a nuclear receptor for dietary lipids. Although LTD(4) is more potent than LTE(4) for inducing calcium flux by the human MC sarcoma line LAD2, LTE(4) is more potent for inducing proliferation and chemokine generation, and is at least as potent for upregulating COX-2 expression and causing prostaglandin D(2) (PGD(2)) generation. LTE(4) caused phosphorylation of extracellular signal-regulated kinase (ERK), p90RSK, and cyclic AMP-regulated-binding protein (CREB). ERK activation in response to LTE(4), but not to LTD(4), was resistant to inhibitors of phosphoinositol 3-kinase. LTE(4)-mediated COX-2 induction, PGD(2) generation, and ERK phosphorylation were all sensitive to interference by the PPARgamma antagonist GW9662 and to targeted knockdown of PPARgamma. Although LTE(4)-mediated PGD(2) production was also sensitive to MK571, an antagonist for the type 1 receptor for cys-LTs (CysLT(1)R), it was resistant to knockdown of this receptor. This LTE(4)-selective receptor-mediated pathway may explain the unique physiologic responses of human airways to LTE(4) in vivo.

More Than One Way to Signal

Although defined by the ability to activate heterotrimeric guanine nucleotide-binding proteins (G proteins), G protein-coupled receptors (GPCRs) can also signal through G protein-independent pathways. Sun et al . used mouse embryo fibroblasts lacking various proteins to investigate the mechanistic relation between G protein-dependent and -independent activation of extracellular signal-regulated kinase (ERK) in response to stimulation of the β 2 -adrenergic receptor (β 2 -AR). Analysis of ERK activation in response to various concentrations of the β 2 -AR agonist isoproterenol revealed a biphasic response. ERK activation in response to low concentrations of isoproterenol was lost in cells lacking Gα s , whereas ERK activation in response to high concentrations was maintained (both required expression of the β 2 -AR). Moreover, ERK activation in response to high concentrations of agonist did not depend on other heterotrimeric G proteins or on β-arrestins. In contrast, low concentrations of isoproterenol stimulated ERK activation in cells lacking Src-family tyrosine kinases, but high concentrations failed to increase the response. Isoproterenol activated Src even in cells lacking both Gα s and β-arrestins. In vitro analysis indicated that Src bound to and phosphorylated the C-terminal tail of the β 2 -AR; further, c-Src coimmunoprecipitated with the β 2 -AR from human embryonic kidney (HEK) 293 cells. When expressed in cells lacking the β 2 -AR, a β 2 -AR mutant that could not bind Src activated Gα s and ERK in response to low concentrations of isoproterenol but failed to elicit enhanced activation of ERK in response to high concentrations. Thus, the authors conclude that low concentrations of agonist activate ERK through a Gα s -mediated pathway, whereas high concentrations turn on a second, Src-dependent, pathway to ERK activation; they speculate that coupling to these two pathways depends on distinct conformations of the β 2 -AR . Y. Sun, J. Huang, Y. Xiang, M. Bastepe, H. Jüppner, B. K. Kobilka, J. J. Zhang, X.-Y. Huang, Dosage-dependent switch from G protein-coupled to G protein-independent signaling by a GPCR. EMBO J. 26 , 53–64 (2007). [PubMed]

Sphingosylphosphorylcholine induces apoptosis of endothelial cells through reactive oxygen species‐mediated activation of ERK

Sphingosylphosphorylcholine (SPC) produces reactive oxygen species (ROS) in MS1 pancreatic islet endothelial cells. In the present study, we explored the physiological significance of the SPC-induced ROS generation in endothelial cells. SPC induced cell death of MS1 cells at higher than 10 microM concentration through a caspase-3-dependent pathway. SPC treatment induced sustained activation of an extracellular signal-regulated kinase (ERK), in contrast to transient activation of ERK in response to platelet-derived growth factor (PDGF)-BB, which stimulated proliferation of MS1 cells. Both the SPC-induced cell death and ERK activation were abolished by pretreatment of the cells with the MEK inhibitor U0126 or by overexpression of a dominant negative mutant of MEK1 (DN-MEK1). Pretreatment of the cells with N-acetylcysteine, an antioxidant, completely prevented the SPC-induced ROS generation, apoptosis, and ERK activation, whereas the ROS generation was not abrogated by treatment with U0126. Consistent with these results, SPC induced cell death of human umbilical vein endothelial cells (HUVECs) through ROS-mediated activation of ERK. These results suggest that the SPC-induced generation of ROS plays a crucial role in the cell death of endothelial cells through ERK-dependent pathway.

Depolarization Activates ERK and Proline-rich Tyrosine Kinase 2 (PYK2) Independently in Different Cellular Compartments in Hippocampal Slices

In the hippocampus, extracellular signal-regulated kinase (ERK) and the non-receptor protein proline-rich tyrosine kinase 2 (PYK2) are activated by depolarization and involved in synaptic plasticity. Both are also activated under pathological conditions following ischemia, convulsions, or electroconvulsive shock. Although in non-neuronal cells PYK2 activates ERK through the recruitment of Src-family kinases (SFKs), the link between these pathways in the hippocampus is not known. We addressed this question using K(+)-depolarized rat hippocampal slices. Depolarization increased the phosphorylation of PYK2, SFKs, and ERK. These effects resulted from Ca(2+) influx through voltage-gated Ca(2+) channels and were diminished by GF109203X, a protein kinase C inhibitor. Inhibition of SFKs with PP2 decreased PYK2 tyrosine phosphorylation dramatically, but not its autophosphorylation on Tyr-402. Moreover, PYK2 autophosphorylation and total tyrosine phosphorylation were profoundly altered in fyn-/- mice, revealing an important functional relationship between Fyn and PYK2 in the hippocampus. In contrast, ERK activation was unaltered by PP2, Fyn knock-out, or LY294002, a phosphatidyl-inositol-3-kinase inhibitor. ERK activation was prevented by MEK inhibitors that had no effect on PYK2. Immunofluorescence of hippocampal slices showed that PYK2 and ERK were activated in distinct cellular compartments in somatodendritic regions and nerve terminals, respectively, with virtually no overlap. Activation of ERK was critical for the rephosphorylation of a synaptic vesicle protein, synapsin I, following depolarization, underlining its functional importance in nerve terminals. Thus, in hippocampal slices, in contrast to cell lines, depolarization-induced activation of non-receptor tyrosine kinases and ERK occurs independently in distinct cellular compartments in which they appear to have different functional roles.

Molecular Mechanisms of Low Intensity Pulsed Ultrasound in Human Skin Fibroblasts

Soluble factors such as polypeptide growth factors, mitogenic lipids, inflammatory cytokines, and hormones are known regulators of cell proliferation. However, the effect of mechanical stimuli on cell proliferation is less well understood. Here we examined the effect of low intensity pulsed ultrasound (US), which is used to promote wound healing, on the proliferation of primary human foreskin fibroblasts and the underlying signaling mechanisms. We show that a single 6-11-min US stimulation increases bromodeoxyuridine incorporation. In addition, an increase in the total cell number is observed after sequential US stimulation. US induced stress fiber and focal adhesion formation via activation of Rho. We further observed that US selectively induced activation of extracellular signal-regulated kinase (ERK) 1/2. Inhibition of Rho-associated coiled-coil-containing protein kinase (ROCK) prevented US-induced ERK1/2 activation, demonstrating that the Rho/ROCK pathway is an upstream regulator of ERK activation in response to US. Consequently, activation of ROCK and MEK-1 was required for US-induced DNA synthesis. Finally, an integrin beta(1) blocking antibody as well as a RGD peptide prevented US-induced DNA synthesis. In addition, US slightly increased phosphorylation of Src at Tyr(416), and Src activity was found to be required for ERK1/2 activation in response to US. In conclusion, our data demonstrate for the first time that US promotes cell proliferation via activation of integrin receptors and a Rho/ROCK/Src/ERK signaling pathway.

Interaction of the tyrosine phosphatase SHP-2 with Gab2 regulates Rho-dependent activation of the c-fos serum response element by interleukin-2.

Gab2 (Grb2-associated binder-2), a member of the IRS (insulin receptor substrate)/Gab family of adapter proteins, undergoes tyrosine phosphorylation in response to cytokine or growth factor stimulation and serves as a docking platform for many signal transduction effectors, including the tyrosine phosphatase SHP-2 [SH2 (Src homology 2)-domain-containing tyrosine phosphatase]. Here, we report that, following IL-2 (interleukin-2) stimulation of human T lymphocytes, SHP-2 binds tyrosine residues 614 and 643 of human Gab2 through its N- and C-terminal SH2 domains respectively. However, the sole mutation of Tyr-614 into phenylalanine is sufficient to prevent Gab2 from recruiting SHP-2. Expression of the Gab2 Tyr-614-->Phe (Y614F) mutant, defective in SHP-2 association, prevents ERK (extracellular-signal-regulated kinase) activation and expression of a luciferase reporter plasmid driven by the c-fos SRE (serum response element), indicating that interaction of SHP-2 with Gab2 is required for ERK activation in response to IL-2. Further investigation of IL-2-dependent induction of SRE showed that expression of a constitutively active mutant of the RhoA GTPase synergizes with IL-2 for SRE-driven transcription, whereas a dominant-negative mutant reduces the IL-2 response. Thus, in response to IL-2, full induction of the SRE requires ERK-dependent as well as Rho-dependent signals that target the Ets-box and the CArG-box respectively. We also report that the synergy between Gab2/SHP-2 and RhoA for IL-2-dependent CArG-box-driven transcription depends upon MEK (mitogen-activated protein kinase/ERK kinase) activation, and is likely to involve regulation of the serum response factor co-activator MAL. Our studies thus provide new insights into the role of Gab2 and SHP-2 in IL-2 signal transduction.

Pseudomonas aeruginosa stimulates phosphorylation of the airway epithelial membrane glycoprotein Muc1 and activates MAP kinase.

We reported previously that Muc1 on the surface of epithelial cells was a receptor for Pseudomonas aeruginosa (Lillehoj EP, Kim BT, and Kim KC. Am J Physiol Lung Cell Mol Physiol 282: L751-L756, 2002). Other studies showed that the Muc1 cytoplasmic tail (CT) contains multiple phosphorylation sites, some of which are phosphorylated constitutively and associated with signaling proteins. However, the relationship between extracellular P. aeruginosa binding and intracellular signaling is unknown. To investigate the signaling mechanism of Muc1, this study examined phosphorylation of its CT and activation of the extracellular signal-regulated kinase (ERK) in response to stimulation by P. aeruginosa or purified flagellin. Our results showed 1) the Muc1 CT was phosphorylated constitutively on serine and tyrosine, 2) serine phosphorylation was stimulated by bacterial cells or flagellin, and 3) binding of P. aeruginosa or flagellin to Muc1 induced phosphorylation of ERK. These results are the first to demonstrate Muc1 CT phosphorylation and ERK activation in response to a clinically important airway pathogen.

Controlling Sprouty Activity

Ligand binding to a receptor tyrosine kinase triggers cascades of intracellular signaling events that regulate numerous cellular processes, such as cell growth and proliferation. To ensure a normal response to growth factors, other proteins are enlisted to limit signaling pathway activity. For example, the conserved membrane-associated protein Sprouty (Spry) antagonizes signaling from various receptor tyrosine kinases by blocking the Ras to extracellular signal-regulated kinase (ERK) pathway. But growth factors also stimulate Spry expression, placing the inhibitor in a negative feedback loop. The circuitry involving Spry is even more complex, as Spry also interacts with other Spry proteins, with receptor-associated adaptor proteins Grb2 and FRS2, and with c-Cbl, an E3-type ubiquitin ligase. Mason et al. report that Spry2 becomes phosphorylated on a conserved tyrosine residue when mammalian fibroblasts are treated with fibroblast growth factor (FGF) or epidermal growth factor (EGF). This modification was necessary for Spry2 to shut down receptor activity. Cells expressing a mutated form of Spry2 lacking the critical tyrosine residue exhibited enhanced ERK activation in response to FGF and EGF. The mutation did not alter membrane localization of Spry2 or its ability to bind to other Spry or adaptor proteins in response to growth factors. However, the mutant was unable to associate with c-Cbl. In the absence of c-Cbl, efficient inhibition of receptor signaling to ERK in response to FGF was observed, indicating that Spry2 does not need to interact with c-Cbl to block receptor signaling. Rather, mutant Spry2 was insensitive to c-Cbl-mediated ubiquitination, indicating that the abundance of Spry2 is likely controlled by c-Cbl. Pharmacologic inhibitors suggest that phosphorylation of Spry2 requires a Src-family kinase. The authors suggest that tyrosine phosphorylation may allow Spry2 to associate with cofactors forming a complex that shuts down receptor signaling to ERK. Proteasomal degradation of Spry2, mediated by c-Cbl, may be a homeostatic control mechanism that is essential for maintaining the positive and negative feedback loops of growth factor receptor signaling circuits. J. M. Mason, D. J. Morrison, B. Bassit, M. Dimri, H. Band, J. D. Licht, I. Gross, Tyrosine phosphorylation of sprouty proteins regulates their ability to inhibit growth factor signaling: A dual feedback loop. Mol. Biol. Cell 15 , 2176-2188 (2004). [Abstract] [Full Text]

Microenvironment regulation of extracellular signal-regulated kinase activity in chondrocytes: effects of culture configuration, interleukin-1, and compressive stress.

To compare extracellular signal-regulated kinase (ERK) activity in response to interleukin-1 (IL-1) in chondrocytes under various culture configurations designed for the study of cartilage biology and repair, and also in response to dynamic load for chondrocytes in cartilage. Isolated bovine articular chondrocytes were maintained in serum-supplemented medium under 4 culture configurations: high-density monolayer, attached to a cut surface of cartilage, within tissue-engineered constructs, or within intact cartilage explants. Samples were subjected to a change of medium with or without IL-1. Cartilage explants were also subjected to dynamic compression. In chondrocyte monolayers, both basal and IL-1-stimulated ERK activities were similarly elevated at 0.5 hours after medium change, diminishing by 74% after 16 hours. In contrast, chondrocytes in other culture configurations exhibited lower basal levels of ERK activity and a moderate activation of ERK in response to IL-1 that was sustained over the 16-hour treatment time. The dynamic component of loading of cartilage explants led to a 5-fold activation of ERK, compared with free-swelling controls, that was indistinguishable from the effects of IL-1. ERK signaling in response to IL-1 in chondrocyte monolayers exhibited a pattern that was distinct from that in other culture systems, suggesting that the extracellular matrix plays an important regulatory role in modulating the response to extracellular stimuli. Since IL-1 and dynamic loading have distinct effects on chondrocyte biosynthesis, signaling pathways other than ERK participate in the chondrocyte responses to these stimuli.

Opposing roles of integrin alpha6Abeta1 and dystroglycan in laminin-mediated extracellular signal-regulated kinase activation.

Laminin-integrin interactions can in some settings activate the extracellular signal-regulated kinases (ERKs) but the control mechanisms are poorly understood. Herein, we studied ERK activation in response to two laminins isoforms (-1 and -10/11) in two epithelial cell lines. Both cell lines expressed beta1-containing integrins and dystroglycan but lacked integrin alpha6beta4. Antibody perturbation assays showed that both cell lines bound to laminin-10/11 via the alpha3beta1and alpha6beta1 integrins. Although laminin-10/11 was a stronger adhesion complex than laminin-1 for both cell lines, both laminins activated ERK in only one of the two cell lines. The ERK activation was mediated by integrin alpha6beta1 and not by alpha3beta1 or dystroglycan. Instead, we found that dystroglycan-binding domains of both laminin-1 and -10/11 suppressed integrin alpha6beta1-mediated ERK activation. Moreover, the responding cell line expressed the two integrin alpha6 splice variants, alpha6A and alpha6B, whereas the nonresponding cell line expressed only alpha6B. Furthermore, ERK activation was seen in cells transfected with the integrin alpha6A subunit, but not in alpha6B-transfected cells. We conclude that laminin-1 and -10/11 share the ability to induce ERK activation, that this is regulated by integrin alpha6Abeta1, and suggest a novel role for dystroglycan-binding laminin domains as suppressors of this activation.

Epidermal Growth Factor Receptor-dependent and -independent Cell-signaling Pathways Originating from the Urokinase Receptor

Urokinase-type plasminogen activator (uPA) and vitronectin activate cell-signaling pathways by binding to the uPA receptor (uPAR). Because uPAR is glycosylphosphatidylinositol-anchored, the signaling receptor is most likely a uPAR-containing multiprotein complex. This complex may be heterogeneous within a single cell and among different cell types. The goal of this study was to elucidate the role of the EGF receptor (EGFR) as a component of the uPAR-signaling machinery. uPA activated extracellular signal-regulated kinase (ERK) in COS-7 cells and in COS-7 cells that overexpress uPAR, and this response was blocked by the EGFR inhibitor, tyrphostin AG1478, implicating the EGFR in the pathway that links uPAR to ERK. By contrast, Rac1 activation, which occurred as a result of uPAR overexpression, was EGFR-independent. COS-7 cell migration was stimulated, in an additive manner, by uPAR-dependent pathways leading to ERK and Rac1. AG1478 inhibited only the ERK-dependent component of the response. CHO-K1 cells do not express EGFR; however, these cells demonstrated ERK activation in response to uPA, indicating the presence of an EGFR-independent alternative pathway. As anticipated, this response was insensitive to AG1478. When CHO-K1 cells were transfected to express EGFR or a kinase-inactive mutant of EGFR, ERK activation in response to uPA was unchanged; however, the EGFR-expressing cells acquired sensitivity to AG1478. We conclude that the EGFR may function as a transducer of the signal from uPAR to ERK, but not Rac1. In the absence of EGFR, an alternative pathway links uPAR to ERK; however, this pathway is apparently silenced by EGFR expression.

A role for Grb2-associated binder-1 in growth hormone signaling.

GH signaling begins with activation of the GH receptor (GHR)-associated cytoplasmic tyrosine kinase, Janus kinase-2. GH-induced Janus kinase-2 activation leads to engagement of several signaling pathways, including the extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase, phosphoinositol 3-kinase, and signal transducer and activator of transcription-5 (STAT5) pathways. Previous work suggests that ERK activation in response to GH may be modulated by several proteins acting as docking molecules, including the epidermal growth factor receptor (EGFR) and insulin receptor substrate-1. In this study we investigate potential roles for the pleckstrin homology (PH) domain-containing insulin receptor substrate-like protein, Grb-2-associated binder-1 (Gab1), in GH signaling. We find in 3T3-F442A preadipocytes that GH promotes tyrosine phosphorylation of Gab1 and its association with SHP2, an Src homology 2-containing cytoplasmic tyrosine phosphatase. The Grb2 adapter protein, in contrast, is specifically coimmunoprecipitated with Gab1, even in the absence of GH exposure. Using a COS-7 cell transient reconstitution system, we observed that GH-induced Gab1 tyrosine phosphorylation is dependent on the Gab1 PH domain, whereas GH-induced coimmunoprecipitation of SHP2 requires tyrosine 627 of Gab1, as previously reported for EGF-induced Gab1-SHP2 association. Deletion of the Gab1 PH domain significantly attenuates GH-induced ERK activation and trans-activation of a c-fos enhancer-driven reporter construct compared with wild-type Gab1 in this system. In contrast, GH-induced STAT5 tyrosine phosphorylation and STAT5-dependent trans-activation are similar in cells expressing wild-type or PH domain-deleted Gab1. Notably, neither the ERK nor the STAT5 GH-dependent signaling outcome is affected by expression of the Gab1 mutant with tyrosine 627 changed to phenylalanine. Finally, we observed GH-dependent translocation of a wild-type, but not a PH domain-deleted, Gab1-green fluorescent protein chimera from the cytoplasm to the plasma membrane. Our results suggest selective involvement of Gab1 in GH-induced ERK activation and implicate the Gab1 PH domain as critical in this involvement.

Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway.

Sprouty (Spry) inhibits signalling by receptor tyrosine kinases; however, the molecular mechanism underlying this function has not been defined. Here we show that after stimulation by growth factors Spry1 and Spry2 translocate to the plasma membrane and become phosphorylated on a conserved tyrosine. Next, they bind to the adaptor protein Grb2 and inhibit the recruitment of the Grb2-Sos complex either to the fibroblast growth factor receptor (FGFR) docking adaptor protein FRS2 or to Shp2. Membrane translocation of Spry is necessary for its phosphorylation, which is essential for its inhibitor activity. A tyrosine-phosphorylated octapeptide derived from mouse Spry2 inhibits Grb2 from binding FRS2, Shp2 or mouse Spry2 in vitro and blocks activation of the extracellular-signal-regulated kinase (ERK) in cells stimulated by growth factor. A non-phosphorylated Spry mutant cannot bind Grb2 and acts as a dominant negative, inducing prolonged activation of ERK in response to FGF and promoting the FGF-induced outgrowth of neurites in PC12 cells. Our findings suggest that Spry functions in a negative feedback mechanism in which its inhibitor activity is controlled rapidly and reversibly by post-translational mechanisms.