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Report: High-Throughput
Mapping of a Dynamic Signaling
Network in Mammalian Cells
Miriam Barrio-Rodiles and Kevin R. Brown et. al.
Science Vol 307 Mar 11 2005
Present by Alex Lei
10/3/2007
Introduction
 Why Dynamic protein-protein interaction network (PPI)?
 Understand the protein functions
 Understand the formations of the protein complexes
 Understand the signal transduction pathways
 Dictate the timing and intensity of network outputs
 Most systematic mapping technology focuses on building
static PPIs in simple organisms e.g. C. elegans, D.
melanogaster etc.
 Develop an automatic high-throughput method
 Systematically map PPIs in mammalian cells
 Ability to construct dynamic PPI
Method
Known as LUMIER (luminescence-based
mammalian interactome mapping)
Three components:
Bait  Protein of interest fused with Renilla
luciferase enzyme (RL)
Prey  mammalian cells with flag-tagged
partners
Antibody  use antibody against flag to create
precipitates of the protein complex
(immunoprecipiates)
Method (cont’d)
Experiment Overview
 Experiment focus --- cell signaling of TGFβ
superfamily
 Growing factor in metazoans (multi-cell
organisms)
Skin cells (Healing wounds)
Bone cells (Bone formations)
Regulates epithelial-to-mesenchymal transition process
(EMT)
 Extracellutar molecules that triggers a series of
processes
Experiment Overview (cont’d)
Signal pathway of TGFβ
Experiment # 1

Purpose: verify the protein post-translation modifications (PTMs)
in Samd pathway using LUMIER

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
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Regulates the dynamics of PPI network to control signal
transduction
Bait  Smad4 (Smad4-RL)
Prey  Flag-Smad2 (2SA)
Findings
1.
Association between Smad4-RL &
Flag-Smad2 with TGFβ signal
2.
Association between TGFβ Type I
Receptor & Smad2 with TGFβ signal
Similar finding also appears
when swapping the bait and prey
(mutants)
Experiment # 2

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Purpose: map the TGFβ PPI network automatically
Method:
 Baits  core members of the pathway with RL-tagged (total 23, some with different
conditions)
 Preys  3 x flag-tagged cDNAs from the FANTOM1 library (total 518)
 Each protein is expressed in the mammalian cells
 Total about 12,000 LUMIER experiments

 Robotic platform to perform automated LUMIER
 Measure by LUMIER intensity ratio (LIR) --- # of fold changes over the control
LIR cutoff = 3
 False-negative 36%
 False-positive 20%
Experiment # 2 (cont’d)
 Resulting static network
 is scale-free network (power law degree distribution)
 Has possible hierarchical modularity
clustering coefficient
Experiment # 2 (cont’d)
Resulting dynamic network
Interactions between Smad2 and Smad4
With absence/presence of TGFβ signals
The movie
Experiment # 3
 Purpose: Identify novel connections with the TGFβ pathway
 Method:
 Apply clustering techniques on the TGFβ LUMIER dataset
 Called binary tree-structured vector quantization (BTSVQ)
 K means clustering
 Self-organizing map
Baits
Prays
Repeated 2 means clustering  binary tree structure
SOM
Experiment # 3 (cont’d) ---background
 K means clustering
 Partition data into K clusters
 Randomized initialization for K class
centroid
 Assign each item to the nearest centroid
 For each class 1 to K
Calculate the centroid
Calculate distance from centroid to each item
Assign each item to the nearest centroid
 Repeat until no items are re-assigned
(convergence) or another stop criterion is
met
K=3
Experiment # 3 (cont’d) ---background
 SOM
 The SOM works both as a
projection (Visualization) method
and a clustering method
 SOM is a neural network approach
that uses an unsupervised training
algorithm through a process called
self-organizing.
 Maps high-dimensional input data
onto a low dimensional (usually
two-dimensional) output space
while preserving the topological
relationships between the input
data
Experiment # 3 (cont’d)
 Results
 PAK1 and TGFβ fall into the same cluster (with similar SOM
patterns)
 PAK family involves in regulating cytoskeletal dynamics, cell
motility, survival and proliferation
 No physical association with TGFβ pathway components have
been reported
 Further investigation on the clustering results show that PAK1binding protein may relate to Occludin (OCLN)
 OCLN is a tight junction accessory protein that is associated with
the cell polarity network
 Verify the interaction between the TGFβ receptors and the PAK1,
OCLN is needed
Experiment # 3 (cont’d)
 By doing a set of experiments on the mammary gland epithelial cells
(NMuMG)
 Discovered OCLN interacts with type I and II receptors with
TGFβ signal
 Discovered OCLN helps the localization of type I receptor
 Located the interacting region of OCLN using LUMIER
(extracellular loop 2)
 Summaries from previous experiments,
 OCLN regulates type I receptor localizations to tight junctions
 Vital to the TGFβ-dependent dissolution of tight junctions during
epithelial-to-mesenchymal transition (EMT)
 Both OCLN and PAK1 regulates TGFβ pathway
Conclusion
 Develop an automated high-throughput
technology to map PPI systematically in
mammalian cells
 Disadvantages
Cannot measure the concentration of the flag-tagged
preys in high-throughput LUMIER
Prone to noise and false positive when the LIR is low
 Discover novel linkage between OCLN, PAK1
and TGFβ in the regulatory pathway
Reference

High-Throughput Mapping of a Dynamic Signaling Network in Mammalian
Cells Miriam Barrios-Rodiles, Kevin R. Brown, Barish Ozdamar, Rohit Bose,
Zhong Liu, Robert S. Donovan, Fukiko Shinjo, Yongmei Liu, Joanna Dembowy,
Ian W. Taylor, Valbona Luga, Natasa Przulj, Mark Robinson, Harukazu Suzuki,
Yoshihide Hayashizaki, Igor Jurisica, and Jeffrey L. Wrana
Science 11 March 2005 307: 1621-1625

Transcriptional control by the TGF- /Smad signaling system Joan Massagué and
David Wotton EMBO Journal Vol 19 No 8 pp 1745-1754, 2000

Lecture slides from Alexander Weiss

Lecture slides from Professor Zhang