December, 2004, Vol.4, No.3

AfCS Reaches Out to Signaling Community

Since the Alliance for Cellular Signaling is first and foremost an experiment in collaborative science, we have pioneered a number of new venues as we seek to communicate with, contribute to, and work with the signaling research community. We are excited by the prospect that key questions about cellular information processing can be answered by a collective approach and that such understanding will produce new paradigms to guide further studies. We invite you to learn more about these

novel efforts, resources, and opportunities. At the annual meeting in May, Al Gilman spoke about three major goals for the AfCS. The first two efforts, the single/double ligand screens and the FXM project, were described in the July 2004 AfCS Newsletter. The third major goal addresses the increasing involvement of the AfCS in the dissemination of information and resources for the enhancement of the signaling research community.

NEWS OF THE

ALLIANCE FOR

CELLULAR SIGNALING

While data are the primary product of the AfCS, we are working hard to reach out to colleagues by supplying a variety of products (1) to inform others of our progress and (2) to facilitate individual research efforts. Equally important, the AfCS is also striving (3) to increasingly engage colleagues in this endeavor, thereby extending this collaborative effort to the signaling research community. We describe some of these efforts in this newsletter.

Hiroaki Kitano (Kitano Symbiotic Systems Project) and Mel Simon (CalTech) discuss the model-based analysis of calcium in macrophage cells.

1. The AfCS Informs the Signaling

Community about our Progress via . . .

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• The AfCS-Nature Signaling Gateway Launched in December 2002, the AfCS-Nature Signaling Gateway (http://www.signaling-gateway.org) is a unique collaboration between the AfCS and the Nature Publishing Group (NPG) and is our primary interface with the research community. The Signaling Update section is compiled and written by Nature editors and provides summaries of recent articles and a library of important papers, reviews, news, jobs, and conferences. In the Molecule Pages, Data Center, and About Us sections, the AfCS provides a public repository of primary data, resources, and information about the AfCS and NPG. In September 2003, the AfCS-Nature Signaling Gateway won the Association of Learned and Professional Society Publishers (ALPSP) Award for Publishing Innovation. Use of the site continues to grow. Since its launch two years ago, the site has grown continuously, and currently NPG sends weekly updates to more than 100,000 unique active addresses. The site has more than 100,000 registered users, and there are more than 100,000 weekly page views. See a presentation on these issues prepared by Timo Hannay of NPG for the May 2004 AfCS annual meeting. The Signaling Gateway represents a new community-driven approach to scientific research. The AfCS uses the site to disseminate and update our own vast collection of experimental data and invites the whole research community to contribute to the process of analysis and discovery with these data. At the same time, invited experts from all over the world contribute to the Molecule Page database to share their knowledge of the individual molecules and pathways that make up the elaborate signaling mechanisms of cells. The entire research community is encouraged to make use of the results in their own investigations and publications. AfCS scientists have no

ity to access the data. prior .

• The News of the Alliance for Cellular Signaling The AfCS Newsletter is written three or four times yearly, posted on the Signaling Gateway, and distributed directly to all AfCS participants (investigators, sponsors, and Molecule Page authors). Currently a link to the newsletter is also distributed by NPG to 100,000 people who signed up for its Signaling Update e-alert which includes 8,000 registered users of the Signaling Gateway who have specifically

indicated their interest in receiving news of the AfCS.

NEWS OF THE

ALLIANCE FOR

CELLULAR SIGNALING

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• The AfCS Research Reports and Brief Communications

The AfCS Research Reports are an important complement to the public accessibility of data collected within our laboratories. Reports describe the purpose, design, and scope of specific experiments and provide the user with a basis for interpreting data. Research Reports and Brief Communications are edited and reviewed by the AfCS and are presented as Web-based publications that are similar in format to articles in traditional scientific journals. However, unlike most traditional journals, the AfCS Research Reports are not meant to highlight “trendy” research from the scientific community. Their purpose is to inform and facilitate the use of AfCS data.

2. The AfCS Facilitates Individual Research

Efforts with . . .

• Molecule Pages (link)

Network Map: A visual overview of the various known states of adenylyl cyclase type 2 and the transitions between them.

The Molecule Page database is a highly structured summary of key facts about proteins involved in mammalian cellular signaling. It covers nearly 4,000 proteins involved in signaling reactions. For each of these, the database currently provides a large amount of automated data collected from numerous other online resources and is updated monthly. These data include names, synonyms, sequence information, biophysical properties, domain and motif information, protein family details, structure and gene data, the identities of orthologs and paralogs, and BLAST results. We provide Mini Molecule Page overviews for 800 proteins that have been composed by expert authors invited by Editorial Board Chair Pat Casey of Duke University. Currently, these authors have completed nearly 100 full-length Molecule Pages that have been forwarded to the Nature Publishing Group for a formal peer review. Several of these have been published on the Signaling Gateway website and more will appear soon.

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Full Molecule Pages include:

• Summary: A written overview of the main characteristics of the molecule, together with key citations and links to PubMed.

• Network Map: A visual overview of the various known states of the molecule and the transitions between them. Different states are defined by, for example, posttranslational modifications of the native peptide, the binding of ligands, the existence of this protein in various complexes with other proteins, or the location of the protein in different subcellular compartments. Clicking on the ovals or arrows of the network map reveals more information about each state and transition, respectively.

• States: A comprehensive list of all known states of the protein, plus relevant citations. Each state has a systematic name that unambiguously identifies it, as well as an author-entered description that is less formal but may be more intuitive to readers. Clicking on a state name provides further details of that state. Clicking on a transition graph icon provides a visual summary of other neighboring states to and from which this state is directly related.

• Transitions: A comprehensive list of all the transitions that occur between known states of the molecule, including information about catalysis of these transitions and relevant citations. Clicking on the name of the transition provides additional details.

• Functions: A comprehensive list of the known biological functions of each state of the molecule. Functions include such roles as enzymatic catalysis and ion transport. Where appropriate, functional attributes such as kinetic parameters and ion permeabilities are also provided, as are relevant citations.

We invite all registered users of the Signaling Gateway to browse the Protein List and search and view the Molecule Page database. The Molecule Pages are a new genre of scientific publication that combines the reliability of peer review with the flexibility and power of an online database. Molecule Pages will be updated annually by authors and are formally citable using digital object identifiers (DOIs).

EXPLORE Published Molecule Pages:

• Adenylyl cyclase type 5 (link) • Acyl protein thioesterase 1 • Adenylyl cyclase type 2 • G protein alpha s • Protein farnesyltransferase, beta

subunit • G protein alpha 11 • Protein N-myristoyltransferase 1 • Abcc6 • Rgs2 • H-Ras • Myt1 • P2Y14 receptor • Ric8a

If you are not currently a registered user, registration can be accomplished quickly and easily at no cost. Registrants at Nature.com have the option of using their user names and passwords from that site.

As the participation of the scientific community is critical to the success of the Molecule Pages effort, we also invite you to go to the author application page to apply to author a Molecule Page. The Membership and Editorial Office (afcsed@mc.duke.edu) is always open to suggestions for additional molecules to add to the Protein List and for nominations of appropriate Molecule Page authors. Together, these efforts will provide

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vital tools for the analysis and reconstruction of cellular signaling networks and thereby deliver an invaluable resource for the signaling community.

• Plasmid Database/Reagents/ATCC

>2700constructs available

through ATTC

Using Invitrogen Gateway technology, the AfCS has generated over 5000 DNA constructs from the combination of 1500 cloned, sequence-verified gene sequences and 200 unique parent vectors. The latter range from vectors for tagging proteins with GFP variants and epitopes to lentiviral vectors used for expression of short hairpin RNAs (shRNAs). All AfCS constructs are recorded in a detailed Web-based plasmid database. This is mirrored at a publicly accessible site that displays those constructs that are available from the American Type Culture Collection (ATCC). Presently, more than 2700 AfCS mouse cDNA clones are available from the ATCC. We invite you to browse the list of plasmid constructs, gather information about the clones, and obtain ATCC ordering information.

The AfCS is utilizing RNA interference (RNAi) to manipulate signaling protein expression in the RAW 264.7 macrophage-like cell line. A set of AfCS-developed RNAi vectors is available from the ATCC. A detailed document entitled A Vector Set For Plasmid-Based RNAi Studies in Mammalian Cells, which describes the vectors and how they can be used for RNAi studies, can be downloaded.

• Antibody Database Results from AfCS testing of over 300 commercially available preparations of antibodies are tabulated in a searchable, interactive database posted on the Web site. To date, more than 135 proteins have been targeted with antibodies assessed by the AfCS Antibody Lab. An AfCS Brief Communication entitled Introduction to the AfCS Antibody Database: Tabulation of Our Experience with Commercially Available Antibodies can be downloaded; this document instructs readers on how to navigate the database and provides information on how the AfCS judges the utility of the antibodies we test.

>300

antibodies

The AfCS Antibody Lab routinely tests the utility of antibodies by Western immunoblotting of whole cell lysates. The information in the current database, which is periodically updated, is accumulated mostly from antibodies that are specific for

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phosphorylated epitopes as part of an effort to quantify ligand-induced changes in protein phosphorylation. A nonexclusive collaboration with Cell Signaling Technology has provided many of the antibodies that have been tested thus far. The AfCS Antibody Lab has combined some of the best phosphospecific antibodies for multiplex Western blotting, which has been employed in ligand screens conducted by the AfCS. These results are available online in the Data Center of the Signaling Gateway Web site. We invite you to explore this valuable resource. Searches for antibodies can be

customized by choosing from options of 21 different vendors, 5 levels of antibody effectiveness, phosphospecific or conventional antibodies, 11 different cell types, and over 75 different ligand treatments of cells.

>135

targets

.

• Phosphoprotein Database

Proteomic analysis of signaling proteins has been carried out by the AfCS Protein Chemistry Laboratory in B cells, WEHI-231 cells, and RAW 264.7 cells. Results can be found in the AfCS Phosphoprotein Database. Largely, the work has been directed toward identification of phosphoproteins and mapping of phosphorylation sites. Data from the experiments involving identification of phosphoproteins from WEHI-231 cells have been made available in two AfCS Brief Communications, Mar 03 and Jan 04, and in the journal Molecular and Cellular Proteomics. These data have proven useful to the signaling community, and the data have been utilized by the Swiss Institute of Bioinformatics for the annotation of posttranslational modifications in the Swiss-Prot database. The data have also been used by Michael Yaffe's group at MIT to develop a Web-based program called Pattern Explorer for discovering phosphorylation site patterns in sets of protein sequence data. At UT Southwestern in Dallas, the Computational Biology Group has utilized the AfCS data to build a Web resource (GiLL) that resolves LocusLink numbers from GenBank GenInfo identifier numbers and vice versa for the analysis of mass spectrometry data. An additional study reporting the identification of 248 phosphoproteins and 210 phosphorylation sites from RAW 264.7 macrophage cells will be published shortly.

>100 Phosphorylated

Proteins and Phosphorylation

Sites

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• Protocols and Description of Reagents

The Data Center of the Signaling Gateway contains over 450 detailed protocols written by AfCS scientists describing procedures, solutions, ligands, and perturbants used in AfCS laboratories. These protocols permit standardization of procedures across AfCS laboratories in addition to serving as a resource for the signaling community.

>450Protocols

3. The AfCS Engages Others by Sharing . . .

• Ligand Screen and FXM Project Data

The primary product of the AfCS is data. We are rapidly generating and making public large-scale sets of well-controlled, high quality data. In the past two years, extensive single and double ligand screens have been performed with two unique cells—mouse splenic B cells and the RAW 264.7 macrophage line. In B cells, ligand screen data involving 25 ligands have been posted for members of the scientific community to analyze, interpret, and follow leads for further investigation. These experiments measure concentrations of intracellular calcium, cAMP, 8 to 10 phosphoproteins [A] [B], and gene transcription. In RAW 264.7 cells, experiments have measured the effect of 23 ligands in single and dual ligand screens. Data have been released for intracellular calcium, cAMP, 21 phosphoproteins, and secretion of 18 cytokines [A] [B] which were measured using the Bio-Rad Bio-Plex Mouse Cytokine 18-Plex Panel. Curation, analysis, and display of these large data sets pose a significant challenge. Analysis requires the combination

and weighting of data from many types of assays involving diverse data types, variable time scales, breadth of scale, and quality. This transformation involves the adoption of

NEW CYTOKINE

DATA !

Data are made public through the AfCS Data Center (link)

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an analytical framework for the ligand screens based on a dimensionless parameter (s-vector) defined in an n-dimensional space. Details of this type of analysis were discussed at the recent annual meeting and generally described in a past newsletter . An example of such an analysis for RAW 264.7 cells is shown in the figure to the right. These ligand screens provide an estimation of signaling diversity within the systems studied. The further analysis of nonadditivity among ligand pairs provides an estimate of the density of interactions among the ligands. Together, the observations define the scope of and provide an initial guide for future modeling studies. The July AfCS Newsletter summarized highlights of the ligand screen data and links readers to PowerPoint presentations and posters presented at the annual AfCS meeting held in May. The July newsletter issue also presented an overview of the Focus on X Module Project (FXM), which is our initial effort to dissect and model a manageable subset of the signaling network of RAW 264.7 macrophage cells.

The Analytic Methods and Results: Example of a transformation

400 5

fmol

Fold

cha

nge

cAMP

TER

00 30s 1 203 8

minutes0 30s 1 203 8

minutes

Calcium

seconds0 600

120 70

Tpeak 25*Tpeak

S-vector

nM

Nor

mal

ized

BLC

0

25 -25

25 -25

Data from RAW 264.7 cells. Intracellular calcium (normalized) and cAMP (expressed by fold change) concentrations are transformed into a single, unitless scale — “S”.

For the signaling community, these data display and analysis tools serve not only to inform scientists of experimental findings, but they also direct them to data sets where experiments may be downloaded, studied, and used in the work of individual investigators. User-friendly and insightful data displays will make AfCS’s findings an increasingly valuable and stimulating resource.

Novel Double Ligand Screen Cytokine Data

Cytokines and chemokines are essential outputs of activated

receptors and signaling networks in macrophages and are critical regulators of inflammatory and immune responses. In part of the dual ligand screen, RAW 264.7 cells were exposed to 22 ligands individually and to each of their possible pair-wise combinations. The results confirm known pathways of cytokine production and reveal novel regulation and interactions between signaling pathways. The cytokine data [A] [B], which are available to the research community to mine and interpret freely, will be integrated by the AfCS with other initiatives (transcriptional microarrays, cAMP, calcium, phosphoproteins, lipids, and RNAi perturbations) to help define and model the complex signaling networks and molecular interactions that regulate cytokine production during infection and inflammation.

Now Available

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• Yeast 2-Hybrid Screen Data To produce a large body of information on protein-protein interactions in our chosen cell types, the AfCS has established a collaboration with Myriad Genetics to perform large-scale yeast 2-hybrid (Y2H) screens. Screening of a Y2H activation domain library of proteins that is representative of a specific cell type may permit the

identification of cell type–specific protein-protein interactions. Currently, Myriad is screening all baits against three libraries. The first library is a new Y2H activation domain library generated from a mixture of three macrophage sources, including resting RAW 264.7 cells, LPS/γ-interferon–stimulated RAW 264.7 cells, and primary bone marrow–derived macrophages. The other two libraries that we chose to continue to screen are the AfCS’s B cell library and a mouse embryo library that has been used extensively by Myriad. This year, 74 baits were selected for screening. Of these, 46 are new baits while 28 are baits chosen for rescreening against the new macrophage library. From these 74 proteins, 514 individual bait fragments were designed by Myriad. The link to the archive of the latest data release provides a text file of all the interactions (currently over

1400), which can be viewed using spreadsheet software. A file compatible with the freely available network mapping program Cytoscape is provided to users, along with instructions on how to view the interaction data with this software.

>115BAITS

>600 PREYS

• RAW 264.7 Image Data The AfCS Microscopy Lab is assessing the cellular location of signaling proteins by dual fluorescence microscopy. Yellow fluorescent protein (YFP)-tagged protein constructs are being expressed in RAW 264.7 cells and their location determined by correlation with coexpressed cyan fluorescent protein (CFP)-tagged subcellular marker proteins. Expression of two complementary fusion constructs that place the fluorescent tags on either the N- or C-terminus of the target protein is being employed. Image data currently available address concerns that addition of the

The translocations of the YFP-tagged Akt PH domains, from cytosol to plasma membrane, serve as biosensors for assaying levels of PIP3 in living cells.

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fluorescent tags might misplace amino- or carboxyl-terminal signals for covalent modification that direct membrane localization. Examples of interesting N- versus C-terminus data sets currently searchable in the Image Database are Lyn, Pak6, Raf1, and Gads. For the FXM effort, the AfCS Microscopy Lab has initiated a single-cell assay using PH domain translocation as the measure of PIP3 responses in RAW 264.7 cells. Upon cellular stimulation by the FXM ligand C5A, PIP3 levels rise, and a YFP-tagged Akt-PH domain (expressed following transfection) translocates from the cytosol to the plasma membrane. It has been found that PH domains from Akt1, Akt2, Akt3, Gab1, Gab2, Itk, CTH2, CTH3, Bam32, and SH3BP2 all translocate clearly from the cytosol to the plasma membrane. In contrast, the PLC delta1 PH domain translocates clearly from the plasma membrane to the cytosol. Data examining 73 PH domains in WEHI-231 cells and RAW 264.7 cells are completed and searchable in the Image Database. To better understand signaling in RAW 264.7 cells, the AfCS Microscopy Lab is assaying responses in single cells in the presence of chemical perturbants and in shRNA-expressing RAW 264.7 cell lines produced by the AfCS Macrophage Lab. Both diminution and enhancement of the peak calcium and PIP3 levels have been observed in response to these perturbations. More subtle features such as the percent of cells responding and the variation in individual time courses can only be detected with single cell measurements. This work is currently ongoing in the AfCS Microscopy Lab.

• Lipidomics Data Sets

An overview of lipidomics procedures showing lipid extraction, identification using mass spectrometry, and an excerpt of a lipid array displaying the results from the statistical analysis of the mass spectral data available in the lipidomics data sets.

A goal of the AfCS Lipidomics Laboratory is to better understand contextual lipid changes in biological membranes following cell signaling events. Currently it is focusing on the analysis of a number of ligands that activate RAW 264.7 macrophages. A

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computational lipidomic approach to this complex problem couples electrospray mass spectrometric analysis with a sophisticated computational method to facilitate the analysis of hundreds (i.e., 450 to date) of lipid species in mammalian cells. This lipid profiling permits identification of temporal changes in membrane lipid species (see figure), which leads to a better understanding of the biophysical and biochemical processes that mediate membrane signaling pathways. The primary function of the Lipidomics Lab in support of the FXM is the generation of lipid arrays after ligand stimulation under various experimental conditions. Lipid arrays generated by stimulation of RAW 264.7 cells by the FXM ligands UDP or C5a or activation of the FcgR1 receptor can be found in the lipidomics data sets currently on the Web site. The effects of UDP in RAW 264.7 cells are likely mediated by P2Y2 and P2Y6 receptors. At present, the AfCS Lipidomics Lab’s analysis of changes in lipid composition in the RAW 264.7 cells is the most comprehensive description of lipid changes known and will be the foundation of the lipid portion of the AfCS modeling effort.

• Microarray Data Sets The Molecular Biology Laboratory has used DNA microarray technology to analyze the transcriptional changes that occur after the addition of ligands to mouse splenic B cells and macrophages. Most recently, an analysis of the major patterns in gene expression from the B cell work is being published by Zhu et al. in the Journal of Immunology (173:12) on December 15, 2004. AfCS B cell ligand screen data are also analyzed in a paper being published in the December 21, 2004 issue of PNAS (101:51) by a non-AfCS group (de Bivort et al.). Data have also been collected examining the effects of the addition of lipopolysaccharide (LPS), a substance known to trigger the innate immune response and activate the macrophage cells. The ordered and selected

expression has been found to correspond well to the known regulatory elements that have been defined for the control of transcription in the early activation of the macrophage. Furthermore, the subset of cytokines and the timing of their transcription conformed well to the AfCS cytokine release data. Current work in the AfCS Molecular Biology Lab is pursuing indications of pathway interactions derived from the AfCS ligand screen and determining the nature of the interactions in double ligand experiments at the level of regulation of transcript formation.

• Modeling Tools The newly created Data Modeling and Network Analysis (DMNA) Laboratory, directed by Adam Arkin at The University of California at Berkley, seeks to build model-based tools to facilitate extraction and testing of hypotheses about how signals propagate in the large-scale signaling networks of interest to the AfCS. The model building and analysis

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tools developed as part of this project are open source. The models developed, the data they are tested against, and the results of model prediction will also be distributed in standardized formats. These models and the tools to manipulate them will allow other scientists interested in the dynamics of signal transduction networks to explore their own hypotheses and use the tools for their own projects.

• Hypothesis Center Rapidly accumulating AfCS data increasingly reveal unexpected patterns, phenotypes, and effects, many of which open avenues to interesting hypotheses and experiments that exceed the capacities of AfCS laboratories. The AfCS is planning a Hypothesis Center that will attempt to promote exploration of AfCS data by the scientific community. The center will initially seek discussion among AfCS laboratory scientists and participating investigators. Each hypothesis will suggest an explanation for a specific AfCS finding, along with experiments designed to test the

explanation. The ultimate goal will be to open this to the general scientific community, whose members will be invited to propose and comment on hypotheses and to present appropriate references or data that they feel support or refute the relevant hypotheses. Anyone will be free to test these hypotheses. SPONSORS

We acknowledge our sponsors with gratitude: The National Institutes of Health

The National Institute of General Medical Sciences The Alliance Project was conceived under the NIGMS Glue Grant Initiative. See: www.nigms.nih.gov/funding/gluegrants.html The National Institute of Allergy and Infectious Diseases The National Cancer Institute

The Pharmaceutical Industry Eli Lilly and Co. The Merck Genome Research Institute Aventis Pharmaceuticals, Inc. Johnson & Johnson It is notable that these corporate sponsors support the investigative aims of the AfCS in compliance with our policy on Intellectual Property.

Philanthropic Foundations: The Agouron Institute Anonymous Foundation, Dallas, TX Others: The University of Texas Southwestern Medical Center We have established collaborative relationships with the following entities: Myriad Genetics, Inc. Cell Signaling Technology Agilent Technologies Isis Pharmaceuticals, Inc. Bio-Rad Laboratories, Inc. These relationships involve either (1) collaborative research agreements in which services are performed for the AfCS at cost or below (with full and prompt disclosure of data) or (2) substantial reductions in pricing for equipment and/or reagents with the hope that successful use of these technologies by the AfCS (and promulgation of such data) will encourage widespread adoption of relevant technologies.

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