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20 publications were found
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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 closePublicationJ Biol Chem, 282(14):10576-84 [ View the Publication] AbstractRegulation 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 closePublicationAnalytical Chemistry, 79:263-272 [ View the Publication] AbstractDiacylglycerols (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 closePublicationMethods Mol Biol., 365:261-86 [ View the Publication] AbstractThe 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 closePublicationMolecular and Cellular Proteomics, 0:M600437-MCP200 [ View the Publication] AbstractCellular 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
- Pubmed (not yet available)
- Data sets
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Zhu X., M. S. Chang, R.Hsueh, R.Taussig, K. D. Smith, P. Sternweis, M. I. Simon and S. Choi. (2006) Dual ligand stimulation of RAW 264.7 cells uncovers feedback mechanisms that regulate TLR-mediated gene expression.. Abstract/Links closePublicationJournal of Immunology, 177:4299-4310 [ View the Publication] AbstractTo characterize how signaling by TLR ligands can be modulated by non-TLR ligands, murine RAW 264.7 cells were treated with LPS, IFN-{gamma}, 2-methyl-thio-ATP (2MA), PGE2, and isoproterenol (ISO). Ligands were applied individually and in combination with LPS, for 1, 2, and 4 h, and transcriptional changes were measured using customized oligo arrays. We used nonadditive transcriptional responses to dual ligands (responses that were reproducibly greater or less than the expected additive responses) as a measure of pathway interaction. Our analysis suggests that cross-talk is limited; <24% of the features with significant responses to the single ligands responded nonadditively to a dual ligand pair. PGE2 and ISO mainly attenuated, while 2MA enhanced, LPS-induced transcriptional changes. IFN-{gamma} and LPS cross-regulated the transcriptional response induced by each other: while LPS preferentially enhanced IFN-{gamma}-induced changes in gene expression at 1 h, IFN-{gamma} signaling primarily attenuated LPS-induced changes at 4 h. Our data suggest specific cross-talk mechanisms: 1) LPS enhances the expression of IFN-{gamma}- response genes by augmenting STAT1 activity and by activating NF-{kappa}B, which synergizes with IFN-{gamma}-induced transcriptional factors; 2) IFN-{gamma} attenuates the late LPS transcriptional response by increasing the expression of suppressor of cytokine signaling 1 and cytokine-inducible SH2-containing protein expression; 3) 2MA modulates LPS secondary transcriptional response by increasing IFN-beta and inhibiting IL-10 gene expression; 4) PGE2 and ISO similarly regulate the LPS transcriptional response. They increase IL-10 transcription, resulting in attenuated expression of known IL-10-suppressed genes. Links
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Shin, K.-J., E. A. Wall, J. R. Zavzavadjian, L. A. Santat, J. Liu, J.-I. Hwang, R. Rebres, T. Roach, W. Seaman, M. I. Simon and Fraser, I. D. C. (2006) A Single Lentiviral Vector Platform for miRNA-Based Conditional RNAi and Coordinated Transgene Expression.. Abstract/Links closePublicationProc Natl Acad Sci U S A, 103:13759-64 [ View the Publication] AbstractRNAi is proving to be a powerful experimental tool for the functional annotation of mammalian genomes. The full potential of this technology will be realized through development of approaches permitting regulated manipulation of endogenous gene expression with coordinated reexpression of exogenous transgenes. We describe the development of a lentiviral vector platform, pSLIK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naive cell system. In mouse embryonic fibroblasts, the pSLIK platform was used to conditionally deplete the expression of the heterotrimeric G proteins Galpha12 and Galpha13 both singly and in combination, demonstrating the Galpha13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of Gbeta2 correlated with a reduced Ca(2+) response to C5a. Insertion of a GFP transgene upstream of the Gbeta2 microRNA-like short hairpin RNA allowed concomitant reexpression of a heterologous mRNA during tetracycline-dependent target gene knockdown, significantly enhancing the experimental applicability of the pSLIK system.
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Jamie A Lee, Robert S Sinkovits, Dennis Mock, Eva L Rab, Jennifer Cai, Peng Yang, Brian Saunders, Robert C Hsueh, Sangdun Choi, Shankar Subramaniam, Richard H Scheuermann, In collaboration with the Alliance for Cellular Signaling (2006) Components of the antigen processing and presentation pathway revealed by gene expression microarray analysis following B cell antigen receptor (BCR) stimulation. Abstract/Links closePublicationBMC Bioinformatics, 7:237 [ View the Publication] AbstractActivation of naive B lymphocytes by extracellular ligands, e.g. antigen, lipopolysaccharide (LPS) and CD40 ligand, induces a combination of common and ligand-specific phenotypic changes through complex signal transduction pathways. For example, although all three of these ligands induce proliferation, only stimulation through the B cell antigen receptor (BCR) induces apoptosis in resting splenic B cells. In order to define the common and unique biological responses to ligand stimulation, we compared the gene expression changes induced in normal primary B cells by a panel of ligands using cDNA microarrays and a statistical approach, CLASSIFI (Cluster Assignment for Biological Inference), which identifies significant co-clustering of genes with similar Gene Ontology annotation. Links
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Pradervand S, Maurya MR, Subramaniam S. (2006) Identification of signaling components required for the prediction of cytokine release in RAW 264.7 macrophages.. Abstract/Links closePublicationGenome Biology, 7(2):R11 [ View the Publication] AbstractRelease of immuno-regulatory cytokines and chemokines during inflammatory response is mediated by a complex signaling network. Multiple stimuli produce different signals that generate different cytokine responses. Current knowledge does not provide a complete picture of these signaling pathways. However, using specific markers of signaling pathways, such as signaling proteins, it is possible to develop a 'coarse-grained network' map that can help understand common regulatory modules for various cytokine responses and help differentiate between the causes of their release. Links
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 Natarajan, M., P. C. Sternweis, K.-M. Lin, R. C. Hsueh, The Alliance for Cellular Signaling Laboratories and R. Ranganathan. (2006) A global analysis of cross-talk in a mammalian cellular signalling network. Abstract/Links closePublicationNature Cell Biology, 8:571-580 [ View the Publication] AbstractCellular information processing requires the coordinated activity of a large network of intracellular signalling pathways. Cross-talk between pathways provides for complex nonlinear responses to combinations of stimuli, but we know little about the density of such interactions in any specific cell. Here, we report the analysis of a large-scale survey of pathway interactions carried out by the Alliance for Cellular Signalling (AfCS1, 2) in the RAW 264.7 macrophage. Twenty-two receptor-specific ligands were studied both alone and in all pairwise combinations for Ca2+ mobilization, cAMP synthesis, phosphorylation of many signalling proteins, and for cytokine production. A large number of non-additive interactions are evident that are consistent with known mechanisms of cross-talk between pathways, but many novel interactions are also revealed. A global analysis of cross-talk suggests that many external stimuli converge on a relatively small number of interaction mechanisms to provide for context-dependent signalling. Links |
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Patrick Flaherty, Michael I. Jordan, Adam P. Arkin (2005) Robust Design of Biological Experiments. Abstract/Links closePublicationProceedings of the Neural Information Processing Symposium 2005, : [ View the Publication] AbstractWe address the problem of robust, computationally-efficient design of biological experiments. Classical optimal experiment design methods have not been widely adopted in biological practice, in part because the resulting designs can be very brittle if the nominal parameter estimates for the model are poor, and in part because of computational constraints. We present a method for robust experiment design based on a semidefinite programming relaxation. We present an application of this method to the design of experiments for a complex calcium signal transduction pathway, where we have found that the parameter estimates obtained from the robust design are better than those obtained from an "optimal" design. Links
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Milne S. B., P. T. Ivanova, D. DeCamp, R. C. Hsueh, and H. A. Brown (2005) A targeted mass spectrometric analysis of phosphatidylinositol phosphate species.. Abstract/Links closePublicationJ Lipid Res., 46(8):1796-802 [ View the Publication] AbstractThe development of a new mass spectrometric lipid profiling methodology permits the identification of cellular phosphatidylinositol monophosphate/phosphatidylinositol bisphosphate/phosphatidylinositol trisphosphate (PIP/PIP2/PIP3) species that includes the fatty acyl composition. Using electrospray ionization mass spectrometry, we were able to resolve and identify 28 PIP and PIP2 compounds as well as 8 PIP3 compounds from RAW 264.7 or primary murine macrophage cell extracts. Analysis of PIP profiles after agonist stimulation of cells revealed the generation of differential PIP3 species and permitted us to propose a novel means for regulation and specificity in signaling through PIP3. This is the first reported identification of intact, cellular PIP3 by mass spectral analysis. The ability to analyze the fatty acyl chain composition of signaling lipids initiates new venues for investigation of the processes by which specific polyphosphoinositide species mediate. Links
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Zhu X, Hart R, Chang MS, Kim JW, Lee SY, Cao YA, Mock D, Ke E, Saunders B, Alexander A, Grossoehme J, Lin KM, Yan Z, Hsueh R, Lee J, Scheuermann RH, Fruman DA, Seaman W, Subramaniam S, Sternweis P, Simon MI, Choi S. (2004) Analysis of the major patterns of B cell gene expression changes in response to short-term stimulation with 33 single ligands.. Abstract/Links closePublicationJ Immunol., 2004 Dec 15;173(12):7141-9 [ View the Publication] AbstractWe examined the major patterns of changes in gene expression in mouse splenic B cells in response to stimulation with 33 single ligands for 0.5, 1, 2, and 4 h. We found that ligands known to directly induce or costimulate proliferation, namely, anti-IgM (anti-Ig), anti-CD40 (CD40L), LPS, and, to a lesser extent, IL-4 and CpG-oligodeoxynucleotide (CpG), induced significant expression changes in a large number of genes. The remaining 28 single ligands produced changes in relatively few genes, even though they elicited measurable elevations in intracellular Ca(2+) and cAMP concentration and/or protein phosphorylation, including cytokines, chemokines, and other ligands that interact with G protein-coupled receptors. A detailed comparison of gene expression responses to anti-Ig, CD40L, LPS, IL-4, and CpG indicates that while many genes had similar temporal patterns of change in expression in response to these ligands, subsets of genes showed unique expression patterns in response to IL-4, anti-Ig, and CD40L. Links
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de Bivort B, Huang S, Bar-Yam Y. (2004) Dynamics of cellular level function and regulation derived from murine expression array data.. Abstract/Links closePublicationProc Natl Acad Sci U S A., 2004 Dec 21;101(51):17687-92 [ View the Publication] AbstractA major open question of systems biology is how genetic and molecular components interact to create phenotypes at the cellular level. Although much recent effort has been dedicated to inferring effective regulatory influences within small networks of genes, the power of microarray bioinformatics has yet to be used to determine functional influences at the cellular level. In all cases of data-driven parameter estimation, the number of model parameters estimable from a set of data is strictly limited by the size of that set. Rather than infer parameters describing the detailed interactions of just a few genes, we chose a larger-scale investigation so that the cumulative effects of all gene interactions could be analyzed to identify the dynamics of cellular-level function. By aggregating genes into large groups with related behaviors (megamodules), we were able to determine the effective aggregate regulatory influences among 12 major gene groups in murine B lymphocytes over a variety of time steps. Intriguing observations about the behavior of cells at this high level of abstraction include: (i) a medium-term critical global transcriptional dependence on ATP-generating genes in the mitochondria, (ii) a longer-term dependence on glycolytic genes, (iii) the dual role of chromatin-reorganizing genes in transcriptional activation and repression, (iv) homeostasis-favoring influences, (v) the indication that, as a group, G protein-mediated signals are not concentration-dependent in their influence on target gene expression, and (vi) short-term-activating/long-term-repressing behavior of the cell-cycle system that reflects its oscillatory behavior. Links
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Shu H, Chen S, Bi Q, Mumby M, Brekken DL. (2004) Identification of phosphoproteins and their phosphorylation sites in the WEHI-231 B lymphoma cell line.. Abstract/Links closePublicationMol Cell Proteomics, Mar;3(3):279-86 [ View the Publication] AbstractA major goal of the Alliance for Cellular Signaling is to elaborate the components of signal transduction networks in model cell systems, including murine B lymphocytes. Due to the importance of protein phosphorylation in many aspects of cell signaling, the initial efforts have focused on the identification of phosphorylated proteins. In order to identify serine- and threonine-phosphorylated proteins on a proteome-wide basis, WEHI-231 cells were treated with calyculin A, a serine/threonine phosphatase inhibitor, to induce high levels of protein phosphorylation. Proteins were extracted from whole-cell lysates and digested with trypsin. Phosphorylated peptides were then enriched using immobilized metal affinity chromatography and identified by liquid chromatography-tandem mass spectrometry. A total of 107 proteins and 193 phosphorylation sites were identified using these methods. Forty-two of these proteins have been reported to be phosphorylated, but only some of them have been detected in B cells. Fifty-four of the identified proteins were not previously known to be phosphorylated. The remaining 11 phosphoproteins have previously only been characterized as novel cDNA or genomic sequences. Many of the identified proteins were phosphorylated at multiple sites. The proteins identified in this study significantly expand the repertoire of proteins known to be phosphorylated in B cells. The number of newly identified phosphoproteins indicates that B cell signaling pathways utilizing protein phosphorylation are likely to be more complex than previously appreciated. Links
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Forrester JS, Milne SB, Ivanova PT, Brown HA. (2004) Computational lipidomics: a multiplexed analysis of dynamic changes in membrane lipid composition during signal transduction.. Abstract/Links closePublicationMol Pharmacol., Apr;65(4):813-21 [ View the Publication] AbstractRecent successes in defining the roles of lipids in cell signaling have stimulated greater interest in these versatile biomolecules. Until recently, analysis of these molecules at the species level has required labor-intensive techniques. The development of electrospray ionization mass spectrometry (ESI-MS) has made possible the detection and identification of thermally labile biological molecules, such as phospholipids. The "soft" ionization does not cause extensive fragmentation, is highly sensitive, accurate, and reproducible. Thus, this method is well suited for analyzing a broad range of phospholipids without elaborate chromatographic separation. Evaluating the vast amounts of data resulting from these measurements is a rate-limiting step in the assessment of phospholipid composition, requiring the development and application of computational algorithms for mass spectrometry data. Here we describe computational lipidomics, a novel analytical technique, coupling mass spectrometry with statistical algorithms to facilitate the comprehensive analysis of hundreds of lipid species from cellular extracts. As a result, lipid arrays are generated to indicate qualitative changes that occur in lipid composition between experimental or disease states, similar to proteomic and genomic analyses. This review presents a methodological strategy for using ESI-MS combined with a high-power computational analysis to profile time-dependent changes in cellular phospholipids after the addition of an agonist or to evaluate changes promoted by pathophysiological processes. As an illustration, we describe the methods and approaches used to generate lipid arrays for The Alliance for Cellular Signaling (AfCS). These arrays are contributing to a more complete understanding of the participants of cellular signaling pathways after activation of cell surface receptors. Links
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Ivanova PT, Milne SB, Forrester JS, Brown HA. (2004) LIPID Arrays: New Tools in the Understanding of Membrane Dynamics and Lipid Signaling.. Abstract/Links closePublicationMol Interv., Apr;4(2):86-96 [ View the Publication] AbstractPhospholipids are the structural building blocks of the membrane bilayer, which retains and regulates intra-cellular content. In addition to creating a protective barrier around the cell, lipids modulate membrane trafficking and are themselves precursors of important intracellular signaling molecules. Identification and quantification of these molecular species is essential for a more complete understanding of cell signaling pathways, and more reliable and sensitive methods are needed for determining membrane phospholipid content. Recent improvements in electrospray ionization mass spectrometry have made possible the direct identification of more than 400 phospholipid species from biological extracts of a single cell type. Changes in the cellular concentration of diverse lipids can be determined by analysis of the mass spectra by statistical algorithms. In the future, lipid arrays will be integrated with other high-throughput profiling technologies, and computational lipidomics will expand our understanding of the molecular basis of cellular processes and diseases. Links
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Robert E. Campbell, Oded Tour, Amy E. Palmer, Paul A. Steinbach, Geoffrey S. Baird, David A. Zacharias, and Roger Y. Tsien
(2002) A monomeric red fluorescent protein. Abstract/Links closePublicationProc Natl Acad Sci U S A, 99(12):7877-7882 [ View the Publication] AbstractAll coelenterate fluorescent proteins cloned to date display some
form of quaternary structure, including the weak tendency of
Aequorea green fluorescent protein (GFP) to dimerize, the obligate
dimerization of Renilla GFP, and the obligate tetramerization of the
red fluorescent protein from Discosoma (DsRed). Although the
weak dimerization of Aequorea GFP has not impeded its accep-
tance as an indispensable tool of cell biology, the obligate tet-
ramerization of DsRed has greatly hindered its use as a genetically
encoded fusion tag. We present here the stepwise evolution of
DsRed to a dimer and then either to a genetic fusion of two copies
of the protein, i.e., a tandem dimer, or to a true monomer
designated mRFP1 (monomeric red fluorescent protein). Each sub-
unit interface was disrupted by insertion of arginines, which
initially crippled the resulting protein, but red fluorescence could
be rescued by random and directed mutagenesis totaling 17
substitutions in the dimer and 33 in mRFP1. Fusions of the gap
junction protein connexin43 to mRFP1 formed fully functional
junctions, whereas analogous fusions to the tetramer and dimer
failed. Although mRFP1 has somewhat lower extinction coeffi-
cient, quantum yield, and photostability than DsRed, mRFP1 ma-
tures >10 times faster, so that it shows similar brightness in living
cells. In addition, the excitation and emission peaks of mRFP1, 584
and 607 nm, are 25 nm red-shifted from DsRed, which should
confer greater tissue penetration and spectral separation from
autofluorescence and other fluorescent proteins.
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Participating investigators and scientists of the Alliance for Cellular Signaling (2002) Overview of the Alliance for Cellular Signaling. Abstract/Links closePublicationNature, 420:703-706 [ View the Publication] AbstractThe Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells -- B lymphocytes (the cells of the immune system) and cardiac myocytes -- to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community. Links
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Ting AY, Kain KH, Klemke RL, and Tsien RY (2001) Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells. Abstract/Links closePublicationProc Natl Acad Sci U S A, 98(26):15003-15008 [ View the Publication] AbstractThe complexity and specificity of many forms of signal transduction are widely believed to require spatial compartmentation of protein kinase and phosphatase activities, yet existing methods for measuring kinase activities in cells lack generality or spatial or temporal resolution. We present three genetically encoded fluorescent reporters for the tyrosine kinases Src, Abl, and epidermal growth factor (EGF) receptor. The reporters consist of fusions of cyan fluorescent protein (CFP), a phosphotyrosine binding domain, a consensus substrate for the relevant kinase, and yellow fluorescent protein (YFP). Stimulation of kinase activities in living cells with addition of growth factors causes 20-35% changes in the ratios of yellow to cyan emissions because of phosphorylation-induced changes in fluorescence resonance energy transfer (FRET). Platelet-derived growth factor (PDGF) stimulated Abl activity most strongly in actin-rich membrane ruffles, supporting the importance of this tyrosine kinase in the regulation of cell morphology. These results establish a general strategy for nondestructively imaging dynamic protein tyrosine kinase activities with high spatial and temporal resolution in single living cells. Links
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Zhang J, Ma Y, Taylor SS, Tsien RY. (2001) Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Abstract/Links closePublicationProc Natl Acad Sci U S A., 98(26):14997-15002 [ View the Publication] AbstractThe complexity and specificity of many forms of signal transduction are widely suspected to require spatial microcompartmentation of protein kinase and phosphatase activities, yet current relevant imaging methods such as phosphorylation-specific antibodies or fluorescent peptide substrates require fixation or microinjection and lack temporal or spatial resolution. We present a genetically encoded fluorescent reporter for protein kinase A (PKA) consisting of fusions of cyan fluorescent protein, a phosphoamino acid binding domain (14-3-3tau ), a consensus substrate for PKA, and yellow fluorescent protein. cAMP elevations cause 25-50% changes in the ratios of yellow to cyan emissions in live cells caused by phosphorylation-induced changes in fluorescence resonance energy transfer. The reporter response was accelerated by tethering to PKA holoenzyme and slowed by localization to the nucleus. We demonstrate that deliberate redistribution of a substrate or colocalizing a substrate and PKA can modulate its susceptibility to phosphorylation by the kinase. The successful design of a fluorescent reporter of PKA activity and its application for studying compartmentalized and dynamic modulation of kinases lays a foundation for studying targeting and compartmentation of PKA and other kinases and phosphatases. Links
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The Alliance for Cellular Signaling (AfCS)
