1. Alliance for Cellular Signaling www.signaling-gateway.org 4 th Annual Meeting; Dallas, May 23-26, 2004

2. Announcements You want to see me suffer? Others?

3. This Talk Brief review of goals and organization Transition from primary cells (B cells, myocytes) to the RAW 264.7 macrophage Current status of major projects/goals Issues: capabilities to be strengthened or developed Everything I say will be (or should be) discussed in greater depth as the meeting proceeds I have not stolen anyone’s slides

4. Central Questions of the AfCS: I Question 1: How complex is signal processing in cells? The set of ligands for cellular receptors is the potential combinatorial code of inputs. How much of this input complexity can a cell uniquely decode as outputs? Experiments: Systematic single- and double- (multi?) ligand screens. Classify output responses; determine degree of crosstalk; identify “hotspots” for later quantitative analysis.

5. Central Questions of the AfCS: II Question 2: What is the structure of the whole signaling network? Is the connectivity sparse or dense? Experiments: Wholesale mapping of relevant protein-protein and small molecule-protein interactions.

6. Central Questions of the AfCS: III Question 3: How much does network topology constrain signal processing capability? How much function is specified by the nature of the connections, rather than by the specific biochemical constants of individual activities. Experiments: Perturbation methods; gain and loss of function, coupled with functional assays.

7. Central Questions of the AfCS: IV Question 4: What are the dynamics of the signaling network? Can we visualize how information propagates through the network and emerges as functional activities? Question 5: Can functional modules be abstracted mathematically? Can we make physical models and predict input- output relationships Question 6: Why is the network the way it is? Why have the observed solutions been chosen? What is being optimized?

8. Management Steering Committee –Gilman, chair; Taussig (COO): monthly meetings Macrophage Committee –Bourne, chair; monthly meetings Analysis Group –Simon, chair; monthly meetings Editorial Board –Casey, chair Lab Meetings (AfCS wide); twice/mo., two labs each Ad hoc Groups –e.g., FXM issues

9. External Advisory Committee Joan Brugge, Chair (Harvard) David Botstein (Princeton) Bill Catterall (U. of Washington) Jack Dixon (UC San Diego) Tony Hunter (Salk Institute) Bob Lefkowitz (Duke) Tony Pawson (Samuel Lunenfeld Research Institute) Paul Sternberg (Caltech)

10. Talent: AfCS Laboratories Bioinformatics: Subramaniam (UCSD) –Sinkovits, Ning, Johnson, Saunders Cell Preparation and Analysis: Sternweis (UTSW) –Hseuh, Lin, DeCamp, Ni Macrophage Physiology: Seaman (UCSF) –Roach, Rebres Molecular Biology: Simon (Caltech) –Fraser, Choi, Eversole-Cire Protein Chemistry: M. Mumby (UTSW) –Shu, Brekken Microscopy: Meyer (Stanford) –Chandy, O’Rourke, Verghese, Whalen, Heo, Liou Antibody: S. Mumby (UTSW) –Han, Levitz Lipidomics: Brown (Vanderbilt) –Forrester, Milne, Ivanova

11. Other VIPs Lily Jiang: COO FXM Madhu Natarajan, Michal Ronen, Xiaocui Zhu, Dennis Mock, Jeff Forrestor, Stuart Johnson: Analysis and modeling Gil Sambrano: MOATLily

12. Bridging Projects Data Modeling and Network Analysis; Arkin Monitoring Plasma Membrane Signaling Events by Evanescent Wave (TIRF) Microscopy (Meyer) Genetically Encoded Indicators of Phosphorylation and Protein Interaction (Tsien) Mapping Signal Transduction Pathways in Living Cells Using PCAs (Michnick) Former BP: Lipidomics (Brown)

13. Collaborations Nature Publishing Group (Signaling Gateway) Myriad Genetics (Y2H) Cell Signaling Technology (Antibodies) Agilent Technologies (DNA microarrays) Biorad (Bio-Plex; phosphoprotein analysis) Isis Pharmaceuticals (Antisense) ATCC (Plasmid distribution)

14. Census Participating investigators: 41 Ph.D. staff scientists: 30 Programmers, bioinformatics staff: 15 Technicians: 40 Administrative: 6 Total ~ 130

15. Current Sponsors NIGMS (Rochelle Long, Mike Rogers) NIAID (Conrad Mallia) NCI Eli Lilly (AfCS & Signaling Gateway; Alph Bingham) Aventis (Bruce Harris) Merck Johnson and Johnson Anonymous Foundation Genentech (Signaling Gateway)

16. The AfCS Cell Systems Initial decision to work with primary cells –mouse B lymphocytes –mouse cardiac myocytes –retrospectively, choice of two was unwise Risky but worth it; use a surrogate B cell line and attempt to develop a cardiac myocyte line Several issues, but the ultimate ironic shaft from the WEHI-231 cell

17. May/June 2003 Terminate cardiac myocytes Phase out B lymphocytes –Complete single ligand screen (Ca 2+, cyclic AMP, phosphoproteins [11], transcripts) –Complete double ligand screen with Ca 2+ & cyclic AMP Initiate work with RAW 264.7 macrophage

18. What has happened since? In a few Words: Last year I came with major problems This year I come with major progress and praise

19. The RAW264.7 Macrophage Diploid; mouse Adherent; easily grown Transfectable; RNAi Receptors: GPCRs, TLRs, TKs, ILs, others Downstream responses –Cytokine secretion –Macropinocytosis –Phagocytosis –Chemotaxis

20. Current Status Major Efforts: three –Single/double ligand screens (Monday) Ca 2+, cAMP, phosphoproteins (21), cytokines (18) Glycerophospholipids, transcripts (arrays & PCR) –FXM project: Focus on X Modules (x = Ca 2+ & PIP3) (Tuesday) –Dissemination of information; enhancements for the signaling research community (Tuesday)

21. July-October 2003 Culture conditions; frozen stocks Chromosome number and stability plus stability of responses Define ligand list (25); dose-response & time relationships –Note: ~25 ligands means 25 2 /2 = >300 combinations for double ligand screen Adapt and expand old assays –Ca 2+, phosphoproteins, lipids Add new assays –Cytokines –Macropinocytosis

22. Ligand Screen November 2003-May 2004 Single/double ligand screen with Ca 2+ and cyclic AMP essentially completed Single/double ligand screen with phosphoproteins (21) 50% complete Single/double ligand screen with cytokines one month from completion Extensive data on glycerophospholipid composition and ligand-induced changes Transcript changes with some ligands and combinations

23. FXM Project October 2003-May 2004 Focus on a module (or two): Ca 2+ & PIP3 –Dissect a portion of the network; crawl before we walk and run; integrate all technologies and analysis Maps: cartoons and Pathway Builder (computational) Parts List: Legacy data; arrays and RT-PCR Assays: populations and single-cells: Ca 2+, PIP3, phosphoproteins (but), lipids Perturbations (the key) –Drugs –RNAi: 41 targeted cell lines (34 targets); more at four/week; I gratefully pay my debt

24. Contributions to the Research Community Website (www/signaling-gateway.org) (NPG) Data –Ligand screen and FXM –Phosphoproteins; phosphorylation sites –Images; locations/translocations of protein & domain families Plasmid database/Reagents/ATCC Yeast Two-Hybrid (Myriad) Antibody Database Protocols Research Reports Molecule Pages (NPG) Newsletter

25. Issues: Ligand Screen Finish it and realign internal resources Primary data: Ca 2+, cAMP, phosphoproteins, cytokines: will be complete “now” and “in December” Other data (transcripts and lipids): experiments will be guided by primary data; full coverage not possible; we have to snoop around Curation and analysis of data: ongoing –Internal debate/inconsistency on extent of analysis –Profiling of ligands; extent of interactions; selection of interacting ligands; placement of biosensors –Pathways and mechanisms; magical inductionism

26. Issues: FXM and Beyond Phenotypes: are they “real”? –Variability, adaptation, selection, off-target hits –If real, …… –Tension: explore phenomena of interest now or later? Multiple knock-downs Scope; when and how to expand; ooze or jump? Additional assays: biosensors and protein phosphorylation Data analysis and modeling: integration of disparate types of data, perturbations, pathway data (Y2H etc.), modeling

27. Issues: Contributions to the Research Community We provide a great deal, but awareness and use can always be improved. –Listen to Timo Hannay (Tuesday) Data is our primary product –Increasing indications of analysis by others What will help? –Publications –“Advertisement”: can we have a story in Nature at the proper moment? –Specific outreach to the Immunology community What else can we monitor? –ATCC; specific information on page hits; data downloads?

28. Issues: Molecule Pages A great deal of talent and effort has gone into this –Has taken significantly longer than expected –Further enhancements are needed, especially a facile search function Idealistic pipedream or major contribution? –The next several months will tell; positive feedback –Truly valuable only if reasonably comprehensive Authorship of a MP is a serious effort; so is authorship of a scholarly review article The carrot: Nature; link to lab web site; others? The stick?

29. Issues: Renewal Application Deadline: October 2004 Cooperation is essential; everyone has to do their job magnificently! Recall the thorny funding problem: we operate on a $10M budget with a $5M cap from NIGMS on awards for glue grants