The FXM Signaling Network

PI(3,4,5)P3 Ca2+

IgG2a

UTP/UDP

C5a

TestingGround

Define the system for study

Plan and organize experiments Reference tool Template for future modeling

?

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?

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?

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?

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?

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.

We built an initial crude network...

…to establish a basic framework

Initial perturbation targets(test method w/ the obvious)

Parts List

More comprehensive map

Y2H targets

FXM Parts List

Started with an inclusive list based on legacy Considered all possible isoforms

Compiled in Excel Worksheets

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

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)

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

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

Pathway Description

Process-based diagrams

Process could be...

Enzymatic Reaction Regulated Reaction Binding interaction Unknown mechanism

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

Protein Non-Protein Complex

Sample Pathway

Putting it all togetherGPCR

Calcium

Fc Receptor

PIP3

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!

Parts List

Pathway Map

Experiments

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