The FXM Signaling Network

2. The FXM Signaling Network UTP/UDP IgG2a C5a Testing Ground PI(3,4,5)P3 Ca2+

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. Parts List More comprehensive map Initial perturbation targets (test method w/ the obvious) 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 Complex Protein Non-Protein

21. Sample Pathway

22. PIP3 GPCR Fc Receptor Calcium Putting it all together

23. Overview of Map • Contains 273 nodes • Connected by 234 processes (reactions) • Complexes constitute 123 nodes • Some molecules such as Lyn, PIP3, or Ca2+ might participate in as many as 15 distinct processes. • Not yet finished!

24. Experiments Parts List Pathway Map

25. 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 Credits