SEARCH · PubMed Health

Results for “Signaling”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 307 records · Page 17Linked to original sources

PubMed2003

The activation of an extracellular signal-regulated kinase by oestradiol interferes with the effects of trastuzumab on HER2 signalling in endometrial adenocarcinoma cell lines.

Cellular response to oestradiol stimuli is mediated both by oestrogen receptor (ER) binding to oestrogen response elements (EREs) and by non-nuclear actions like activation of mitogen-activated protein kinase (MAPK) signal transduction. Therefore, oestradiol stimuli might be able to interfere with the action of antitumoral substances directed against receptor tyrosine kinase signalling. We investigated the effect of oestradiol on the inhibition of HER2 signalling by trastuzumab (Herceptin) in two human endometrial adenocarcinoma cell lines. Activation of the extracellular signal-regulated kinase (ERK-1/2), a major mediator of HER2 signalling, was measured by means of western blotting experiments and ERE activation was determined in transient reporter-gene assays. In endometrial Ishikawa and HEC-1A adenocarcinoma cells, HER2 signalling was inhibited by trastuzumab only in the absence of oestradiol. We were able to demonstrate that oestradiol counteracted the inhibitory effects of trastuzumab by rapid phosphorylation of ERK-1/2, a kinase downstream of the HER2 receptor. The pure anti-oestrogen ICI 182,780 was able to restore both the trastuzumab-triggered inhibition of the ERK-1/2 pathway and the antiproliferative action of this substance in Ishikawa cells. Our data suggest that combinations of trastuzumab with anti-oestrogens may be effective in the treatment of endometrial cancers with a positive ER and HER2 receptor status.

Adenocarcinoma↗

PubMed1999

Regulation of Smad signalling by protein associations and signalling crosstalk.

Smads are intracellular signalling mediators for the family of transforming growth factor beta (TGF-beta)-related growth and differentiation factors, which signal through transmembrane serine/threonine kinases. Following receptor-induced activation, heteromeric Smad complexes translocate into the nucleus, where they act as transcription factors. Recent progress has revealed that Smad signalling is not merely determined by activation of the class of TGF-beta receptors, but is also regulated through crosstalk with other kinase signalling cascades. In addition, the Smads regulate transcription through functional cooperativity and physical interactions with other transcription factors, which might also be targets for regulation by other signalling cascades. This signalling crosstalk might explain the complexity of the responses to TGF-beta and related factors.

Animals↗

PubMed2002

Estrogen action and cytoplasmic signaling pathways. Part II: the role of growth factors and phosphorylation in estrogen signaling.

In recent years, distinct signaling pathways involving specific complexes of cytoplasmic proteins have been shown to orchestrate estrogen action. These pathways might supplement or augment genomic effects of estrogen that are attributable to transcriptional activation by liganded receptor. Signals might be transduced through phosphorylation of the estrogen receptors (ERs), or indirectly through effects upon transcriptional coactivators or cell receptors. Estrogen signaling is coupled to growth factor signaling with feedback mechanisms directly impacting function of growth factor receptors. These signaling pathways regulate important physiological processes, such as cell growth and apoptosis. Here, we focus on cytoplasmic signaling pathways leading to activation of ERs.

Animals↗

PubMed1999

The organization of INAD-signaling complexes by a multivalent PDZ domain protein in Drosophila photoreceptor cells ensures sensitivity and speed of signaling.

Phototransduction in Drosophila has emerged as an attractive model system for studying the organization of signaling cascades in vivo. In photoreceptor neurons, the multivalent PDZ protein INAD serves as a scaffold to assemble different components of the phototransduction pathway, including the effector PLC, the light-activated ion channel TRP, and a protein kinase C involved in deactivation of the light response. INAD is required for organizing and maintaining signaling complexes in the rhabdomeres of photoreceptors. This macromolecular organization endows photoreceptors with many of their signaling properties, including high sensitivity, fast activation and deactivation kinetics, and exquisite feedback regulation by small localized changes in [Ca2+]i. Assembly of transduction components into signaling complexes is also an important cellular strategy for ensuring specificity of signaling while minimizing unwanted cross-talk. In this report, we review INAD's role as a signal transduction scaffold and its role in the assembly and localization of photoreceptor complexes.

Animals↗

PubMed1997

Calpain regulation of cytoskeletal signaling complexes in von Willebrand factor-stimulated platelets. Distinct roles for glycoprotein Ib-V-IX and glycoprotein IIb-IIIa (integrin alphaIIbbeta3) in von Willebrand factor-induced signal transduction.

The adhesion of platelets to sites of vascular injury is critically dependent on the binding of subendothelial bound von Willebrand factor (vWf) to the platelet surface glycoprotein complexes, GP Ib-V-IX and GP IIb-IIIa (integrin alphaIIbbeta3). There is growing evidence that the binding of vWf to these receptors is not only essential for stable platelet adhesion but is also important for the transduction of activation signals required for changes in platelet morphology, granule secretion, and platelet aggregation. In this study we have investigated signaling events induced by vWf binding to GP Ib-V-IX in both spreading and aggregated platelets. The adhesion of platelets to vWf resulted in dramatic actin filament reorganization, as assessed by immunofluorescence with fluorescein isothiocyanate-conjugated phalloidin, and the cytoskeletal recruitment of various structural proteins (talin and integrin alphaIIbbeta3) and signaling enzymes (pp60c-src, focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI 3-kinase), and protein-tyrosine phosphatase (PTP)-1B). Time course experiments in both spreading and aggregated platelets revealed that talin, FAK, and PTP-1B were proteolyzed after translocation to the cytoskeleton. The proteolysis of these proteins was dependent on the presence of extracellular calcium and was specifically inhibited by pretreating platelets with the membrane-permeable calpain inhibitors calpeptin, E64d, and MDL 28,170, but not with the membrane-impermeable inhibitors leupeptin, E64, and calpastatin. The cytoskeletal translocation of signaling enzymes in vWf-stimulated platelets was abolished by pretreating platelets with an anti-GP Ib-V-IX antibody but was unaffected by blocking ligand binding to integrin alphaIIbbeta3. In contrast, calpain activation in vWf-stimulated platelets required ligand binding to both GP Ib-V-IX and integrin alphaIIbbeta3. The activation of calpain in both spreading and aggregated platelets resulted in a substantial decrease in the level of tyrosine phosphorylation of multiple platelet proteins and was associated with a 50-80% reduction in the amount of cytoskeletal associated talin, integrin alphaIIbbeta3, PI 3-kinase, FAK, pp60(c-)src, and PTP-1B. These studies suggest a potentially important role for calpain in regulating the formation and/or stability of cytoskeletal signaling complexes in vWf-stimulated platelets. Furthermore, they demonstrate distinct roles for GP Ib-V-IX and integrin alphaIIbbeta3 in vWf-induced signal transduction.

Blood Platelets↗

PubMed1997

Spatial patterns of Ca2+ signals define intracellular distribution of a signaling by Ca2+/Calmodulin-dependent protein kinase II.

Ca2+ plays a central role in cell signaling, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a major mediator of Ca2+ actions. The spatial distribution of intracellular Ca2+ signaling is not homogenous, rather it is dynamically organized, and it has been speculated that spatial patterns of Ca2+ signals may function as a form of cellular information transmitted to downstream molecules. To address this issue, we studied the intracellular distributions of the signalings by CaMKII and Ca2+ in the same astrocytes. The former was visualized by monitoring site-specific phosphorylation of a cytoskeletal protein vimentin, using site- and phosphorylation-specific antibodies, while the latter was examined by fura-2-based Ca2+ microscopy. Local Ca2+ signals induced vimentin phosphorylation by CaMKII localized in the same area. On the other hand, Ca2+ waves in astrocytes induced global phosphorylation of vimentin by CaMKII. A small population of vimentin filaments highly phosphorylated by CaMKII underwent structural alteration into short filaments at electron microscopic level. These results indicate that CaMKII transmits spatial patterns of Ca2+ signals to vimentin as cellular information. The possibility is discussed that spatial patterns of vimentin phosphorylation may be important for intracellular organization of vimentin filament networks.

Actin Cytoskeleton↗

PubMed1997

The hsp90-binding antibiotic geldanamycin decreases Raf levels and epidermal growth factor signaling without disrupting formation of signaling complexes or reducing the specific enzymatic activity of Raf kinase.

We have expressed the mitogenic signaling proteins Src, Ras, Raf-1, Mek (MAP kinase kinase), and Erk (MAP kinase) in baculovirus-infected Sf9 insect cells in order to study a potential role for the chaperone hsp90 in formation of multiprotein complexes. One such complex obtained by immunoadsorption with anti-Ras antibody of cytosol prepared from cells simultaneously expressing Ras, Raf, Mek, and Erk contained Ras, Raf, and Erk. To detect directly the protein-protein interactions involved in forming multiprotein complexes, we combined cytosols from single infections in vitro in all possible combinations of protein pairs. We detected complexes between Ras.Raf, Ras.Src, Raf.Mek, and Raf.Src, but no complex containing Erk was obtained by mixing cytosols. Thus, cellular factors appear to be required for assembly of the Erk-containing multiprotein complex. One cellular factor thought to be involved in signaling protein complex formation is the chaperone hsp90, and we show that Src, Raf, and Mek are each complexed with insect hsp90. Treatment of Sf9 cells with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, did not decrease coadsorption of either Raf or Erk with Ras, although it did decrease the level of cytosolic Raf. To study geldanamycin action, we treated rat 3Y1 fibroblasts expressing v-Raf and showed that the antibiotic blocked assembly of Raf.hsp90 complexes at an intermediate stage of assembly where Raf is still bound to the p60 and hsp70 components of the assembly mechanism. As in Sf9 cells, Raf levels decline with geldanamycin treatment of 3Y1 cells. To determine if geldanamycin affects mitogenic response, we treated HeLa cells with epidermal growth factor (EGF) and showed that geldanamycin treatment decreased EGF signaling and decreased the level of Raf protein without affecting the EGF-mediated increase in Raf kinase activity. We conclude that hsp90 is not required for forming complexes between the mitogenic signaling proteins or for Raf kinase activity and that EGF signaling is decreased indirectly by geldanamycin because the antibiotic increases degradation of Raf and perhaps other components of the signaling pathway.

Animals↗

PubMed1999

Quantitative relationship among integrin-ligand binding, adhesion, and signaling via focal adhesion kinase and extracellular signal-regulated kinase 2.

Because integrin-mediated signals are transferred through a physical architecture and synergistic biochemical network whose properties are not well defined, quantitative relationships between extracellular integrin-ligand binding events and key intracellular responses are poorly understood. We begin to address this by quantifying integrin-mediated FAK and ERK2 responses in CHO cells for varied alpha(5)beta(1) expression level and substratum fibronectin density. Plating cells on fibronectin-coated surfaces initiated a transient, biphasic ERK2 response, the magnitude and kinetics of which depended on integrin-ligand binding properties. Whereas ERK2 activity initially increased with a rate proportional to integrin-ligand bond number for low fibronectin density, the desensitization rate was independent of integrin and fibronectin amount but proportional to the ERK2 activity level with an exponential decay constant of 0.3 (+/- 0.08) min(-1). Unlike the ERK2 activation time course, FAK phosphorylation followed a superficially disparate time course. However, analysis of the early kinetics of the two signals revealed them to be correlated. The initial rates of FAK and ERK2 signal generation exhibited similar dependence on fibronectin surface density, with both rates monotonically increasing with fibronectin amount until saturating at high fibronectin density. Because of this similar initial rate dependence on integrin-ligand bond formation, the disparity in their time courses is attributed to differences in feedback regulation of these signals. Whereas FAK phosphorylation increased to a steady-state level as new integrin-ligand bond formation continued during cell spreading, ERK2 activity was decoupled from the integrin-ligand stimulus and decayed back to a basal level. Accordingly, we propose different functional metrics for representing these two disparate dynamic signals: the steady-state tyrosine phosphorylation level for FAK and the integral of the pulse response for ERK2. These measures of FAK and ERK2 activity were found to correlate with short term cell-substratum adhesivity, indicating that signaling via FAK and ERK2 is proportional to the number of integrin-fibronectin bonds.

Animals↗

PubMed1999

Cholesterol efflux-mediated signal transduction in mammalian sperm. beta-cyclodextrins initiate transmembrane signaling leading to an increase in protein tyrosine phosphorylation and capacitation.

Sperm capacitation in vitro is highly correlated with an increase in protein tyrosine phosphorylation that is regulated by cAMP through a unique mode of signal transduction cross-talk. The activation of this signaling pathway, as well as capacitation, requires bovine serum albumin (BSA) in the incubation medium. BSA is hypothesized to modulate capacitation through its ability to remove cholesterol from the sperm plasma membrane. Here we demonstrate that the cholesterol-binding heptasaccharides, methyl-beta-cyclodextrin and OH-propyl-beta-cyclodextrin, promote the release of cholesterol from the mouse sperm plasma membrane in media devoid of BSA. Both of these beta-cyclodextrins were also demonstrated to increase protein tyrosine phosphorylation in the absence of BSA in both mouse and bull sperm, and the patterns of phosphorylation were similar to those induced by media containing BSA. The potency of the different beta-cyclodextrins to increase protein tyrosine phosphorylation in sperm was correlated with their cholesterol binding efficiencies, and preincubation of the beta-cyclodextrins with cholesterol-SO4- to saturate their cholesterol-binding sites blocked the ability of these compounds to stimulate protein tyrosine phosphorylation. The beta-cyclodextrin effect on protein tyrosine phosphorylation was both NaHCO3 and protein kinase A-dependent. The beta-cyclodextrins were also able to capacitate mouse sperm in the absence of BSA, as measured by the ability of the zona pellucida to induce the acrosome reaction and by successful fertilization in vitro. In summary, beta-cyclodextrins can completely replace BSA in media to support signal transduction leading to capacitation. These data further support the coupling of cholesterol efflux to the activation of membrane and transmembrane signaling events leading to the activation of a unique signaling pathway involving the cross-talk between cAMP and tyrosine kinase second messenger systems, thus defining a new mode of cellular signal transduction initiated by cholesterol release.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed2002

Activator of G-protein signaling 1 blocks GIRK channel activation by a G-protein-coupled receptor: apparent disruption of receptor signaling complexes.

The Ras-related protein, activator of G-protein signaling 1 (AGS1) or Dexras1, interacts with G(i)/G(o)alpha and activates heterotrimeric G-protein signaling systems independent of a G-protein-coupled receptor (GPCR). As an initial approach to further define the cellular role of AGS1 in GPCR signaling, we determined the influence of AGS1 on the regulation of G(betagamma)-regulated inwardly rectifying K(+) channel (GIRK) current (I(ACh)) by M(2)-muscarinic receptor (M(2)-MR) in Xenopus oocytes. AGS1 expression inhibited receptor-mediated current activation by >80%. Mutation of a key residue (G31V) within the G(1) domain involved in nucleotide binding for Ras-related proteins eliminated the action of AGS1. The inhibition of I(ACh) was not overcome by increasing concentrations of the muscarinic agonist acetylcholine but was progressively lost upon injection of increasing amounts of M(2)-MR cRNA. These data suggest that AGS1 may antagonize GPCR signaling by altering the pool of heterotrimeric G-proteins available for receptor coupling and/or disruption of a preformed signaling complex. Such regulation would be of particular importance for those receptors that exist precoupled to heterotrimeric G-protein and for receptors operating within signaling complexes.

Acetylcholine↗

PubMed2002

The role of Grp 78 in alpha 2-macroglobulin-induced signal transduction. Evidence from RNA interference that the low density lipoprotein receptor-related protein is associated with, but not necessary for, GRP 78-mediated signal transduction.

The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds to many proteins, some of which trigger signal transduction. Receptor-recognized forms of alpha(2)-Macroglobulin (alpha(2)M*) bind to LRP, but the pattern of signal transduction differs significantly from that observed with other LRP ligands. For example, neither Ni(2+) nor the receptor-associated protein, which blocks binding of all known ligands to LRP, block alpha(2)M*-induced signal transduction. In the current study, we employed alpha(2)-macroglobulin (alpha(2)M)-agarose column chromatography to purify cell surface membrane binding proteins from 1-LN human prostate cancer cells and murine macrophages. The predominant binding protein purified from 1-LN prostate cancer cells was Grp 78 with small amounts of LRP, a fact that is consistent with our previous observations that there is little LRP present on the surface of these cells. The ratio of LRP:Grp 78 is much higher in macrophages. Flow cytometry was employed to demonstrate the presence of Grp 78 on the cell surface of 1-LN cells. Purified Grp 78 binds to alpha(2)M* with high affinity (K(d) approximately 150 pm). A monoclonal antibody directed against Grp 78 both abolished alpha(2)M*-induced signal transduction and co-precipitated LRP. Ligand blotting with alpha(2)M* showed binding to both Grp 78 and LRP heavy chains in these preparations. Use of RNA interference to silence LRP expression had no effect on alpha(2)M*-mediated signaling. We conclude that Grp 78 is essential for alpha(2)M*-induced signal transduction and that a "co-receptor" relationship exists with LRP like that seen with several other ligands and receptors such as the uPA/uPAR (urinary type plasminogen activator or urokinase/uPA receptor) system.

Animals↗

PubMed2003

Type 1 insulin-like growth factor receptor (IGF-IR) signaling inhibits apoptosis signal-regulating kinase 1 (ASK1).

The type 1 insulin-like growth factor receptor (IGF-IR) is a receptor-tyrosine kinase that plays a critical role in signaling cell survival and proliferation. IGF-IR binding to its ligand, insulin-like growth factor (IGF-I) activates phosphoinositide 3-kinase (PI3K), promotes cell proliferation by activating the mitogen-activated protein kinase (MAPK) cascade, and blocks apoptosis by inducing the phosphorylation and inhibition of proapoptotic proteins such as BAD. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase (MAPKKK) that is required for c-Jun N-terminal kinase (JNK) and p38 activation in response to Fas and tumor necrosis factor (TNF) receptor stimulation, and for oxidative stress- and TNFalpha-induced apoptosis. The results presented here indicate that ASK1 forms a complex with the IGF-IR and becomes phosphorylated on tyrosine residue(s) in a manner dependent on IGF-IR activity. IGF-IR signaling inhibited ASK1 irrespective of TNFalpha-induced ASK1 activation and resulted in decreased ASK1-dependent JNK1 stimulation. Signaling through IGF-IR rescued cells from ASK1-induced apoptotic cell death in a manner independent of PI3K activity. These results indicate that IGF-IR signaling suppresses the ASK-1-mediated stimulation of JNK/p38 and the induction of programmed cell death. The simultaneous activation of MAP kinases and the inhibition of the stress-activated arm of the cascade by IGF-IR may constitute a potent proliferative signaling system and is possibly a mechanism by which IGF-I can stimulate growth and inhibit cell death in a wide variety of cell types and biological settings.

Animals↗

PubMed2004

Caveolar and lipid raft localization of the growth hormone receptor and its signaling elements: impact on growth hormone signaling.

The growth hormone receptor (GHR) is a cell surface receptor that mediates the somatogenic and metabolic effects of the growth hormone (GH). GHR signaling is transduced via the receptor-associated cytoplasmic tyrosine kinase called Janus protein kinase 2 (JAK2). The major intracellular signaling systems activated by JAK2 in response to GH include the signal transducer and activator of transcription (STAT) 5 and extracellular signal-regulated kinase (ERK)-1 and -2 pathways. In this report, we investigate the role of cholesterol-rich plasma membrane microdomains (caveolae and lipid rafts) in GH signaling. By subcellular fractionation of the GH-responsive 3T3-F442A murine preadipocyte, we found dramatic enrichment (6.7-fold) of plasma membrane GHR in the caveolae membranes (CM). JAK2 was also represented in the CM fraction, but was less enriched (2.5-fold) than GHR. ERK1/2 and the important ERK pathway upstream small adaptor protein, Grb2 (growth factor receptor-bound protein 2), were also enriched in caveolae (2.3- and 8.3-fold, respectively), but STAT5 was barely detected in the same fraction. Correspondingly, GH-induced tyrosine-phosphorylated GHR, JAK2, and ERK1/2 were highly represented in the CM fraction, whereas tyrosine-phosphorylated STAT5 was enriched in the non-membranous fraction of the post-nuclear supernatant. Additionally, GH induced further accumulation of GHR, Grb2, and SHC proteins in the CM fraction. Interestingly, treatment of the cells with the caveolae-disrupting agent, methyl-beta-cyclodextrin (mbetaCD), selectively inhibited GH-induced ERK1/2 activation but not STAT5 phosphorylation; repletion of cholesterol in mbetaCD-treated cells restored GH-induced ERK activation. Comparison of 3T3-F442A cells with the GHR-expressing human IM-9 lymphoblasts revealed similar enrichment of GHR in the lipid raft fraction of IM-9 as in the CM fraction of 3T3-F442A, but there were dramatic differences in the ERKs and Grb2. The IM-9 cell, in which ERKs are not activated by GH, displayed no enrichment of ERKs and Grb2 in the lipid raft fraction. Our results suggest that localization of GHRs in the CM fraction of the plasma membrane plays important roles in signaling.

3T3 Cells↗

PubMed2005

Cystic fibrosis airway epithelial Ca2+ i signaling: the mechanism for the larger agonist-mediated Ca2+ i signals in human cystic fibrosis airway epithelia.

In cystic fibrosis (CF) airways, abnormal epithelial ion transport likely initiates mucus stasis, resulting in persistent airway infections and chronic inflammation. Mucus clearance is regulated, in part, by activation of apical membrane receptors coupled to intracellular calcium (Ca(2+)(i)) mobilization. We have shown that Ca(2+)(i) signals resulting from apical purinoceptor (P2Y(2)-R) activation are increased in CF compared with normal human airway epithelia. The present study addressed the mechanism for the larger apical P2Y(2)-R-dependent Ca(2+)(i) signals in CF human airway epithelia. We show that the increased Ca(2+)(i) mobilization in CF was not specific to P2Y(2)-Rs because it was mimicked by apical bradykinin receptor activation, and it did not result from a greater number of P2Y(2)-R or a more efficient coupling between P2Y(2)-Rs and phospholipase C-generated inositol 1,4,5-trisphosphate. Rather, the larger apical P2Y(2)-R activation-promoted Ca(2+)(i) signals in CF epithelia resulted from an increased density and Ca(2+) storage capacity of apically confined endoplasmic reticulum (ER) Ca(2+) stores. To address whether the ER up-regulation resulted from ER retention of misfolded DeltaF508 CFTR or was an acquired response to chronic luminal airway infection/inflammation, three approaches were used. First, ER density was studied in normal and CF sweat duct human epithelia expressing high levels of DeltaF508 CFTR, and it was found to be the same in normal and CF epithelia. Second, apical ER density was morphometrically analyzed in airway epithelia from normal subjects, DeltaF508 homozygous CF patients, and a disease control, primary ciliary dyskinesia; it was found to be greater in both CF and primary ciliary dyskinesia. Third, apical ER density and P2Y(2)-R activation-mobilized Ca(2+)(i), which were investigated in airway epithelia in a long term culture in the absence of luminal infection, were similar in normal and CF epithelia. To directly test whether luminal infection/inflammation triggers an up-regulation of the apically confined ER Ca(2+) stores, normal airway epithelia were chronically exposed to supernatant from mucopurulent material from CF airways. Supernatant treatment expanded the apically confined ER, resulting in larger apical P2Y(2)-R activation-dependent Ca(2+)(i) responses, which reproduced the increased Ca(2+)(i) signals observed in CF epithelia. In conclusion, the mechanism for the larger Ca(2+)(i) signals elicited by apical P2Y(2)-R activation in CF airway epithelia is an expansion of the apical ER Ca(2+) stores triggered by chronic luminal airway infection/inflammation. Greater ER-derived Ca(2+)(i) signals may provide a compensatory mechanism to restore, at least acutely, mucus clearance in CF airways.

Adult↗

PubMed2006

The alliance for cellular signaling plasmid collection: a flexible resource for protein localization studies and signaling pathway analysis.

Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines.

Animals↗

PubMed2005

Post-translational disulfide modifications in cell signaling--role of inter-protein, intra-protein, S-glutathionyl, and S-cysteaminyl disulfide modifications in signal transmission.

Cell signaling entails a host of post-translational modifications of effector-proteins. These modifications control signal transmission by regulating the activity, localization or half-life of the effector-protein. Prominent oxidative modifications induced by cell-signaling reactive oxygen species (ROS) are cysteinyl modifications such as S-nitrosylation, sulfenic acid and disulfide formation. Disulfides protect protein sulfhydryls against oxidative destruction and simultaneously influence cell signaling by engaging redox-regulatory sulfhydryls in effector-proteins. The types of disulfides implicated in signaling span (1) protein S-glutathionylation, e.g. as a novel mode of Ras activation through S-glutathionylation at Cys-118 in response to a hydrogen-peroxide burst, (2) intra-protein disulfides, e.g. in the regulation of the stability of the protein phosphatase Cdc25C by hydrogen-peroxide, (3) inter-protein disulfides, e.g. in the hydrogen peroxide-mediated inactivation of receptor protein-tyrosine phosphatase alpha (RPTPalpha) by dimerization and (4) protein S-cysteaminylation by cystamine. Cystamine is a byproduct of pantetheinase-catalyzed pantothenic acid recycling from pantetheine for biosynthesis of Coenzyme A (CoA), a ubiquitous and metabolically indispensable cofactor. Cystamine inactivates protein kinase C-epsilon (PKCepsilon), gamma-glutamylcysteine synthetase and tissue transglutaminase by S-cysteaminylation-triggered mechanisms. The importance of protein S-cysteaminylation in signal transmission in vivo is evident from the ability of cystamine administration to rescue the intestinal inflammatory-response deficit of pantetheinase knockout mice. These mice lack the predominant epithelial pantetheinase isoform and have sharply reduced levels of cystamine/cysteamine in epithelial tissues. In addition, intraperitoneal administration of cystamine significantly delays neurodegenerative pathogenesis in a Huntington's disease mouse model. Thus, cystamine may serve as a prototype for the development of novel therapeutics that target effector-proteins regulated by S-cysteaminylation.

Animals↗

PubMed1995

Early molecular events induced by T cell receptor (TCR) signaling in immature CD4+ CD8+ thymocytes: increased synthesis of TCR-alpha protein is an early response to TCR signaling that compensates for TCR-alpha instability, improves TCR assembly, and parallels other indicators of positive selection.

Differentiation of immature CD4+ CD8+ thymocytes into mature CD4+ or CD8+ T cells occurs within the thymus and is dependent upon expression of antigen receptor complexes (T cell receptor [TCR]) containing clonotypic alpha/beta proteins. We have recently found that CD4+ CD8+ thymocytes express low levels of surface TCR because of limitations placed on TCR assembly by the instability of nascent TCR-alpha proteins within the endoplasmic reticulum (ER) of immature thymocytes. Because TCR-alpha/beta expression increases during development, a molecular mechanism must exist for increasing the number of assembled TCR complexes present in immature CD4+ CD8+ thymocytes that have been signaled to differentiate into mature T cells, although no such mechanism has yet been described. In the current report we have examined the molecular consequences of intracellular signals generated by engagement of surface TCR complexes on immature CD4+ CD8+ thymocytes. Isolated TCR engagement generated signals that increased TCR-alpha RNA levels and increased synthesis of TCR-alpha proteins, which, in turn, significantly increased assembly of complete TCR-alpha/beta complexes in CD4+ CD8+ thymocytes. Increased TCR-alpha protein levels in TCR-signaled CD4+ CD8+ thymocytes was the result of increased synthesis and not increased stability of TCR-alpha proteins, indicating that TCR engagement compensates for, but does not correct, the inherent instability of TCR-alpha proteins in the ER of immature thymocytes. Consistent with the delivery by TCR engagement of a positive selection signal, TCR engagement also increased CD5 expression, decreased RAG-1 expression, and decreased CD4/CD8 coreceptor expression in immature CD4+ CD8+ thymocytes. These data identify amplified TCR-alpha expression as an initial response of immature CD4+ CD8+ thymocytes to TCR-mediated positive selection signals and provide a molecular basis for increased surface TCR density on developing thymocytes undergoing selection events within the thymus.

Animals↗

PubMed2004

Jasmonate and ethylene signalling and their interaction are integral parts of the elicitor signalling pathway leading to beta-thujaplicin biosynthesis in Cupressus lusitanica cell cultures.

Roles of jasmonate and ethylene signalling and their interaction in yeast elicitor-induced biosynthesis of a phytoalexin, beta-thujaplicin, were investigated in Cupressus lusitanica cell cultures. Yeast elicitor, methyl jasmonate, and ethylene all induce the production of beta-thujaplicin. Elicitor also stimulates the biosynthesis of jasmonate and ethylene before the induction of beta-thujaplicin accumulation. The elicitor-induced beta-thujaplicin accumulation can be partly blocked by inhibitors of jasmonate and ethylene biosynthesis or signal transduction. These results indicate that the jasmonate and ethylene signalling pathways are integral parts of the elicitor signal transduction leading to beta-thujaplicin accumulation. Methyl jasmonate treatment can induce ethylene production, whereas ethylene does not induce jasmonate biosynthesis; methyl jasmonate-induced beta-thujaplicin accumulation can be partly blocked by inhibitors of ethylene biosynthesis and signalling, while blocking jasmonate biosynthesis inhibits almost all ethylene-induced beta-thujaplicin accumulation. These results indicate that the ethylene and jasmonate pathways interact in mediating beta-thujaplicin production, with the jasmonate pathway working as a main control and the ethylene pathway as a fine modulator for beta-thujaplicin accumulation. Both the ethylene and jasmonate signalling pathways can be regulated upstream by Ca(2+). Ca(2+) influx negatively regulates ethylene production, and differentially regulates elicitor- or methyl jasmonate-stimulated ethylene production.

Cells, Cultured↗