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Recent Publications
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Jiang LI, Collins J, Davis R, Lin KM, DeCamp D, Roach T, Hsueh R, Rebres RA, Ross EM, Taussig R, Fraser I, Sternweis PC (2007) se of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway Abstract/Links.
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PublicationJ Biol Chem, 282(14):10576-84 [ View the Publication] Abstract
Regulation of intracellular cyclic adenosine 3 ',5 '-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed Gs-dependent receptors for isoproterenol and prostaglandin E2. Whereas two ligands, uridine 5 '-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via Gq/calcium and Gi, the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for Gs-coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P2 receptor and the heterotrimeric G13 protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP. Links
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Callender HL, Forrester JS, Ivanova P, Preininger A, Milne S, and Brown HA. (2007) Quantification of diacylglycerol species from biological extracts by electrospray ionization mass spectrometry Abstract/Links.
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PublicationAnalytical Chemistry, 79:263-272 [ View the Publication] Abstract
Diacylglycerols (DAGs) play significant roles in both intermediate metabolism and signal transduction. These lipid species are second messengers involved in modulating a plethora of cellular processes. Evaluation of DAG species concentrations has been hampered by the lack of a reliable method for molecular species analysis within a complex mixture of cellular lipids. We describe a new method for quantitative analysis of DAG species from complex biological extracts based on positive mode electrospray ionization mass spectrometry without prior derivatization. Quantification is achieved using internal standards and calibration curves constructed by spiking cell extracts with different concentrations of DAG species containing various acyl chain lengths and degrees of unsaturation. The new mass spectral data processing algorithm incorporates a multiple linear regression model including a factor accountable for possible interactions between experimental preparations and the slope of the curve for the standards, allowing the examinations of the effects of sample origin conditions (such as cell types, phenotypes, etc.) and instrument variability on this slope. Internal standards provide a basis for quantification of 28 DAG molecular species detected in RAW 264.7 cells after stimulation of a G-protein coupled receptor with platelet activating factor. This method displays excellent reproducibility over the established range of concentrations with variations of < or =10% and is highly sensitive with a detection limit of 0.1-0.4 pmol/microL depending upon acyl chain composition. We have shown differential effects on various DAGs in response to a ligand which illustrates the importance of examining lipids at the molecular species level rather than as a single homogeneous entity. Links
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Fraser I, Liu W, Rebres R, Roach T, Zavzavadjian J, Santat L, Liu J, Wall E, Mumby M. (2007) The use of RNA interference to analyze protein phosphatase function in mammalian cells Abstract/Links.
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PublicationMethods Mol Biol., 365:261-86 [ View the Publication] Abstract
The use of RNA interference to knock down protein phosphatases has proven to be a valuable approach to understanding the functions of these enzymes in mammalian cells. Many protein phosphatases exist as multisubunit and multigene families, which has made it difficult to assess their physiological functions using traditional approaches. The ability to selectively knock down specific subunits and individual isoforms with RNA interference has begun to make it possible to determine the contributions of individual phosphatase proteins to cellular signaling. This chapter describes methods for knocking down protein phosphatases with small interfering RNAs in easily transfectable cells and by the introduction of short-hairpin RNAs into less tractable cells using lentivirus vectors. Links
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Zavzavadjian JR, Couture S, Park WS, Whalen J, Lyon S, Lee G, Fung E, Mi Q, Liu J, Wall E, Santat L, Dhandapani K, Kivork C, Driver A, Zhu X, Chang MS, Randhawa B, Gehrig E, Bryan H, Verghese M, Maer A, Saunders B, Ning Y, Subramaniam S, Meyer T, Simon M, O'Rourke N, Chandy G, Fraser ID (2007) The Alliance for Cell Signaling plasmid collection: a flexible resource for protein localization studies and signaling pathway analysis. Abstract/Links.
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PublicationMolecular and Cellular Proteomics, 0:M600437-MCP200 [ View the Publication] Abstract
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 (AfCS), 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 proteomic applications. To date, we have generated over 3,000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at http://www.signaling-gateway.org/data/plasmid/
that allows users to browse, search and blast AfCS 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 proteomic resource for large-scale subcellular localization screens in mammalian cell lines. Links- PDF
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The Alliance for Cellular Signaling (AfCS)
