2. The FXM Signaling Network PI(3,4,5)P3Ca 2+ IgG2a UTP/UDP C5a Testing Ground

3. Define the system for study  Plan and organize experiments  Reference tool  Template for future modeling ?

4. We asked...  What signaling molecules might be involved?  Are these signaling proteins specifically expressed in RAW 264.7 cells?  What is the presumed role of each signaling molecule in the network?  Which signaling proteins shall be targets of perturbations and other experiments?

5. We asked...  What signaling molecules might be involved?  Are these signaling proteins specifically expressed in RAW 264.7 cells?  What is the presumed role of each signaling molecule in the network?  Which signaling proteins shall be targets of perturbations and other experiments?

6. We asked...  What signaling molecules might be involved?  Are these signaling proteins specifically expressed in RAW 264.7 cells?  What is the presumed role of each signaling molecule in the network?  Which signaling proteins shall be targets of perturbations and other experiments?

7. We asked...  What signaling molecules might be involved?  Are these signaling proteins specifically expressed in RAW 264.7 cells?  What is the presumed role of each signaling molecule in the network?  Which signaling proteins shall be targets of perturbations and other experiments?

8. Initial Goals of FXM Project  Develop a parts list of specific proteins that constitute the FXM network.  Develop a detailed map describing known or suspected reactions occurring within FXM pathways.

9. We built an initial crude network... …to establish a basic framework

10. Initial perturbation targets (test method w/ the obvious) Parts List More comprehensive map Y2H targets

11. FXM Parts List  Started with an inclusive list based on legacy  Considered all possible isoforms

12. Compiled in Excel Worksheets

13. Expression in RAW 264.7 Cells  Expression tracked by Affymetrix arrays and RT-PCR  In some cases, antibody data and/or functional data are available  Goal: trim down list

14. Overview of Parts  247 proteins (including isoforms) considered  204 proteins remain on list following expression analysis  ~40 mRNA confirmed present by RT-PCR  ~27 proteins confirmed present by antibody  ~64 expression is uncertain (i.e., Affy unclear, RT not yet done)

15. Building a detailed network map  Collaboration with Adam Arkin Lab  Development of map using PathwayBuilder software (DARPA BioSPICE)  Tested both the method for creating a signaling map and the suitability of the software

16. Content of a Pathway Signaling Map  NOT a connection map of all possible protein interactions A more defined map:  Assign a role for each signaling molecule (protein and non-protein) within the FXM network  Molecular states and complexes  Create a set of hypotheses to be tested by experimental perturbation  A tool and template for modelers

17. Pathway Description

18. Process-based diagrams Process could be...  Enzymatic Reaction  Regulated Reaction  Binding interaction  Unknown mechanism

20. Nodes  Each node represents a specific state of a molecule or group of molecules  Nodes are unique  Nodes are designated as proteins, non-proteins, or complexes ProteinNon-Protein Complex

21. Sample Pathway

22. Putting it all together GPCR Calcium Fc Receptor PIP3

23. Overview of Map  Contains 273 nodes  Connected by 234 processes (reactions)  Complexes constitute 123 nodes  Some molecules such as Lyn, PIP3, or Ca 2+ might participate in as many as 15 distinct processes.  Not yet finished!

24. Parts List Pathway Map Experiments

25. Credits  Lily Jiang  Madhu Natarajan  Alex Gilman  Adam Arkin  Elliott Ross  Al Gilman  Mike Berridge  Henry Bourne  Rick Brown  Lew Cantley  Mike Gold  Kelly Smith  Jim Stull